WO2020238788A1 - 抗肿瘤的联合用药物及其应用 - Google Patents

抗肿瘤的联合用药物及其应用 Download PDF

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WO2020238788A1
WO2020238788A1 PCT/CN2020/091753 CN2020091753W WO2020238788A1 WO 2020238788 A1 WO2020238788 A1 WO 2020238788A1 CN 2020091753 W CN2020091753 W CN 2020091753W WO 2020238788 A1 WO2020238788 A1 WO 2020238788A1
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antibody
her2
tumor
drug
alkyl
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French (fr)
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汤伟佳
马健
梅星星
俞金泉
李胜峰
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百奥泰生物制药股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6851—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a determinant of a tumour cell
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/4745—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems condensed with ring systems having nitrogen as a ring hetero atom, e.g. phenantrolines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5365—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes

Definitions

  • the invention relates to the field of medical biology, in particular to an anti-tumor combined drug and its application.
  • Combination drugs require appropriate drug compatibility, but if the combination drugs are not properly selected, the desired results may not be achieved, or even counterproductive. The main reason is that in the process of combined medication, the drug has a complex reaction in the body, rather than a simple one plus one equals two effect. Choosing a suitable combination of drugs can enhance the efficacy or reduce side effects. However, improper selection of drugs can reduce the efficacy of the drugs or cause unwanted side effects. It has been reported that the current incidence of adverse reactions caused by drug interactions exceeds 30%.
  • the purpose of the present invention is to provide a safe and efficient anti-tumor combination drug.
  • the present invention provides the following technical solutions:
  • a combined anti-tumor drug including anti-HER2-antibody drug conjugate and anti-PD-1 antibody.
  • the anti-HER2-antibody drug conjugate and the anti-PD-1 antibody are separate administration units.
  • the anti-HER2-antibody drug conjugate and the anti-PD-1 antibody together form a combined administration unit.
  • the anti-HER2-antibody drug conjugate is a compound represented by Formula I or a pharmaceutically acceptable salt thereof,
  • Abu is an anti-HER2 antibody, and p is selected from 1-10;
  • X is -H or a halogen group
  • R 2 is -H or C1-C6 alkyl
  • R 4 is -OH or -SH
  • R 5 is C1-C6 alkyl or benzyl
  • R 6 is C1-C6 alkyl, phenyl or benzyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-6 alkyl
  • Z is independently -H or C1-C4 hydrocarbon group, or two Z and the carbon atoms to which they are attached form a carbonyl group;
  • the invention also provides an application of the anti-tumor drug combination as described above.
  • the specific technical solutions are as follows:
  • a method for treating tumors includes: administering an effective dose of anti-PD-1 antibody and anti-HER2-antibody drug conjugate to patients in need of treatment.
  • the present invention has the following beneficial effects:
  • the inventors of the present invention found in research that when the anti-PD-1 antibody and the anti-HER2-antibody drug conjugate are used in combination, the anti-PD-1 antibody and the anti-HER2-antibody drug conjugate can cooperate with each other to achieve synergy. Synergistic effect, improve the effect of suppressing tumors.
  • the anti-HER2-antibody drug conjugate formed by the anti-HER2 antibody and one or more chemical drugs connected to it works together through antibody-dependent cell-mediated cytotoxicity and chemical drugs, and the anti-HER2 antibody can also Realize the targeted delivery of chemical drugs, thus further significantly increasing the killing effect of combined drugs on tumor cells.
  • the anti-PD-1 antibody as an immune check site inhibitor, can stimulate the tumor-killing function of cells, thereby significantly improving the inhibitory effect on tumors.
  • the combined drug of the present invention can improve the inhibitory effect on tumors, and at the same time, tumor patients also have good tolerance, and achieve the purpose of safe and efficient medication.
  • Figure 1 is the inhibitory effect curve of the combination of antibody C and ADC1 and ADC1 alone on the proliferation of SK-BR-3 cells in the presence of T cells;
  • Figure 2 shows the inhibitory effect curve of antibody C and ADC1 in combination and ADC1 alone on the proliferation of NCI-87 cells in the presence of T cells;
  • Figure 3 shows the inhibitory effect curve of the combination of antibody C and ADC1 and ADC1 alone on the proliferation of SK-BR-3 cells in the presence of PBMC;
  • FIG. 4 shows the results of FACS analysis of target antigen (HER2) expression in NCI-87 cells
  • FIG. 5 shows the results of FACS analysis of target antigen (HER2) expression in B16F10-hHER2 cells
  • Figure 6 is a curve of the inhibitory effect of ADC1 on the proliferation of B16F10-hHER2 cells
  • Figure 7 is the inhibitory effect curve of positive drug Cisplatin on the proliferation of B16F10-hHER2 cells
  • Figure 8 is a graph showing the change trend of the effect of the combination of antibody C and ADC1 and ADC1 alone on the tumor volume in mice;
  • Figure 9 is a graph showing the change trend of the effect of the combination of antibody C and ADC1 and ADC1 alone on the body weight of mice.
  • antibody refers to any form of antibody that exhibits a desired biological activity (for example, inhibition of the binding of a ligand to its receptor or inhibition of receptor signal transduction induced by the ligand). Therefore, “antibody” is administered in its broadest sense, and clearly includes but is not limited to monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies.
  • monoclonal antibody in the present invention is also referred to as “monoclonal antibody”, which refers to antibodies obtained from a population of substantially the same antibody, and each antibody constituting the population is identical. Monoclonal antibodies are highly specific and can target a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations which usually include multiple different antibodies directed against multiple different determinants (epitopes), each monoclonal antibody is directed against only a single determinant on the antigen.
  • the expression anti-HER2 antibody (Anti-HER2 antibody) in the embodiment of the present invention refers to an antibody that uses the proto-oncogene human epidermal growth factor receptor 2 (human epidermal growth factor receptor-2, HER2) as the target protein.
  • Anti-PD-1 antibody (Anti-PD-1 antibody) refers to an antibody that uses programmed death-1 (PD-1) as its target protein.
  • antibody-drug conjugate in the present invention refers to a binding protein such as an antibody or its antigen which is chemically linked to one or more chemical drugs (which may optionally be therapeutic or cytotoxic agents) Fragment.
  • the ADC includes an antibody, cytotoxic or therapeutic drug, and a linker that enables the drug to be attached or coupled to the antibody.
  • ADC usually has 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 drugs conjugated to the antibody.
  • the drugs that can be included in ADC include, but are not limited to: mitotic inhibitors, anti-tumor antibiotics, immunomodulators, carriers for gene therapy, alkylating agents, anti-angiogenic agents, antimetabolites, boron-containing reagents, chemotherapy protective agents, Hormones, antihormones, corticosteroids, photoactive therapeutics, oligonucleotides, radionuclide reagents, topoisomerase inhibitors, tyrosine kinase inhibitors, and radiosensitizers.
  • the drug included in the ADC may be a maytansinoid drug.
  • the drug included in the ADC may be a compound represented by Formula I or a pharmaceutically acceptable salt thereof as described in this application.
  • the antibody forms a disulfide bond through its own cysteine or thiolated amino acid, such as thiolated lysine, and is coupled to the drug.
  • alkyl and alkylene as used herein are meant to include branched and straight chain saturated aliphatic hydrocarbon groups having a specified number of carbon atoms.
  • C1-C6 in “C1-C6 alkyl” includes groups having 1, 2, 3, 4, 5, or 6 carbon atoms arranged in a linear or branched chain.
  • C1-C6 specifically includes methyl, ethyl, n-propyl, isopropyl, n-butyl, tert-butyl, isobutyl, pentyl (including 8 isomers), hexyl ( Including 23 kinds of isomers).
  • cycloalkyl refers to a monocyclic saturated aliphatic hydrocarbon group having a specified number of carbon atoms.
  • cycloalkyl includes cyclopropyl, methyl-cyclopropyl, 2,2-dimethyl-cyclobutyl, 2-ethyl-cyclopentyl, cyclohexyl, and the like.
  • halogen as used herein includes fluorine, chlorine, bromine and iodine.
  • amino acid side chain refers to a substituent that replaces a certain group (such as a hydrogen atom) in an amino acid; for example, a glycine side chain is a substituent formed by replacing a hydrogen atom on a glycine methylene group.
  • amino acid side chains include, but are not limited to, natural amino acid side chains.
  • administering and “treatment” are used to refer to animals, humans, experimental subjects, cells, tissues, organs, or biological fluids, it means to combine exogenous drugs, therapeutic agents, diagnostic agents or compositions with animals, humans, and recipients. Contact with the treated person, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, treatment methods, pharmacokinetic methods, diagnostic methods, research methods, and experimental methods. Treating cells includes contacting the agent with the cell and contacting the agent with a fluid, where the fluid contacts the cell.
  • administering and “treatment” also mean treatment of cells in vitro and ex vivo, for example, by reagents, diagnostic agents, binding compositions, or by other cells.
  • “Inhibition” as used herein includes delaying the development of symptoms associated with a disease and/or reducing the severity of these symptoms that the disease will or expected to develop. The term also includes alleviating existing symptoms, preventing additional symptoms, and alleviating or preventing the underlying causes of these symptoms. Therefore, the term means that beneficial results have been given to vertebrate subjects suffering from diseases.
  • therapeutically effective amount refers to when the anti-HER2-antibody drug conjugate (anti-HER2-ADC) and/or anti-PD-1 antibody or fragments thereof are administered to cells, tissues or In the subject, it effectively prevents or slows the amount of the disease or condition to be treated.
  • a therapeutically effective dose further refers to the amount of the antibody drug conjugate (ADC) and/or antibody or fragment thereof that is sufficient to cause alleviation of symptoms, such as treating, curing, preventing or alleviating related medical conditions, or improving the symptom.
  • the treatment rate, cure rate, prevention rate or slowdown rate of the disease are examples of the disease.
  • the effective amount for a specific subject may vary depending on various factors, such as the disease to be treated, the overall health of the patient, the method of administration and dosage, and the severity of side effects.
  • the effective amount can be the maximum dose or dosing schedule that avoids significant side effects or toxic effects.
  • the therapeutically effective amount refers to the ingredient alone.
  • the therapeutically effective amount refers to the combined amount of active ingredients that produce a therapeutic effect, regardless of whether it is administered in combination, continuous administration, or simultaneous administration.
  • a therapeutically effective amount will reduce symptoms usually by at least 10%; usually at least 20%; at least about 30%; at least 40% or at least 50%.
  • Anti-HER2-antibody drug conjugates or anti-PD-1 antibody antibodies can form a wide variety of pharmaceutically acceptable salts, including but not limited to: acid addition salts formed with organic acids, such organic acids include but not Limited to aliphatic monocarboxylic and dicarboxylic acids, phenyl-substituted alkanoic acid, hydroxyalkanoic acid, alkanedioic acid, aromatic acid, aliphatic and aromatic sulfonic acid, amino acid, etc., such as acetic acid, trifluoroacetic acid, propionic acid , Glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid , Salicylic acid, etc.; acid addition salt
  • the term "individual”, “subject” or “patient” as used herein refers to an animal that is the target of treatment, observation, or experiment.
  • the patient may be (but not limited to) a mammal, including (but not limited to) a human.
  • the present invention provides a combined anti-tumor drug, including: anti-HER2-antibody drug conjugate (anti-HER2-ADC) and anti-PD-1 antibody;
  • anti-HER2-ADC anti-HER2-antibody drug conjugate
  • anti-PD-1 antibody anti-PD-1 antibody
  • the anti-HER2-antibody drug conjugate and the anti-PD-1 antibody are separate administration units.
  • or the anti-HER2-antibody drug conjugate and the anti-PD-1 antibody together form a combined administration unit.
  • the anti-HER2-antibody drug conjugate and the anti-PD-1 antibody are separate administration units. This way, on the one hand, the stability of anti-HER2-ADC and anti-PD-1 antibodies when stored separately can be improved; on the other hand, in actual use, the dosage of anti-HER2-ADC and anti-PD-1 antibodies can be flexibly adjusted.
  • the anti-HER2-ADC and anti-PD-1 antibodies can be administered simultaneously, sequentially or separately.
  • the anti-PD-1 antibody may be administered before the anti-HER2-ADC is administered, and/or after the anti-HER2-ADC is administered, and/or while the anti-HER2-ADC is administered. Therefore, the anti-HER2-ADC and anti-PD-1 antibodies are preferably separate administration units, which are convenient to be administered to patients with HER2-positive cancer.
  • Combined administration means that the anti-HER2-ADC and anti-PD-1 antibodies are administered in the same treatment cycle so that the patient has an effective dose of anti-HER2-ADC and anti-PD-1 antibodies at the same time. ;
  • the combined administration can be that the anti-HER2-ADC and anti-PD-1 antibodies are administered on the same day or on different days. It can also be administered at the same time when the actual operation permits.
  • the anti-HER2-antibody drug conjugate is a compound represented by Formula I or a pharmaceutically acceptable salt thereof,
  • Abu is an anti-HER2 antibody, and p is selected from 1-10;
  • X is -H or a halogen group
  • R 2 is -H or C1-C6 alkyl
  • R 4 is -OH or -SH
  • R 5 is C1-C6 alkyl or benzyl
  • R 6 is C1-C6 alkyl, phenyl or benzyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-6 alkyl
  • Z is independently -H or C1-C4 hydrocarbon group, or two Z and the carbon atoms to which they are attached form a carbonyl group;
  • R 11 is selected from -H, alkyl, aryl, cycloalkyl, cycloalkenyl, heteroaryl and heterocycle, or two R 11 and nitrogen form a heterocycle, wherein one carbon in the heterocycle can be selected Or two oxygen substitutions.
  • the anti-HER2-antibody drug conjugate is a compound represented by Formula II or a pharmaceutically acceptable salt thereof:
  • Abu is an anti-HER2 antibody, and p is selected from 1-10;
  • X is -H or -Cl
  • Y is -H or methyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-C6 alkyl
  • the anti-HER2-antibody drug conjugate is a compound represented by Formula III or a pharmaceutically acceptable salt thereof:
  • Abu is an anti-HER2 antibody, and p is selected from 1-10;
  • X is -H or -Cl
  • Y is -H or methyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-C6 alkyl
  • n is selected from any integer from 1-20. m is preferably 1-10, more preferably 5-10.
  • the anti-HER2-antibody drug conjugate is a compound represented by Formula IV or a pharmaceutically acceptable salt thereof:
  • Abu is an anti-HER2 antibody
  • p is selected from 1-10.
  • the anti-HER2 antibody in the anti-HER2-antibody drug conjugate is one of Antibody A, Antibody B, Trastuzumab (Trastuzumab) and Pertuzumab (Pertuzumab) or Multiple
  • the amino acid sequence of the light chain of antibody A is shown in SEQ ID NO. 1, and the amino acid sequence of the heavy chain of antibody A is shown in SEQ ID NO. 2; or the light chain or heavy chain sequence of antibody A has one amino acid Or multiple substitutions, but the biological activity remains unchanged.
  • the amino acid sequence of the light chain of antibody B is shown in SEQ ID NO. 3, and the amino acid sequence of the heavy chain of antibody B is shown in SEQ ID NO. 4; or the light chain or heavy chain sequence of antibody B has one or more amino acids A replacement, but the biological activity remains unchanged.
  • the drug in the anti-HER2-antibody drug conjugate, can be coupled to the anti-HER2 antibody Abu via a linker.
  • the structure after the drug and the linker are connected is selected from the compound described in formula V, VI, VII, or VIII:
  • X is -H or a halogen group
  • R 2 is -H or C1-C6 alkyl
  • R 4 is -OH or -SH
  • R 5 is C1-C6 alkyl or benzyl
  • R 6 is C1-C6 alkyl, phenyl or benzyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-6 alkyl
  • Z is independently -H or C1-C4 hydrocarbon group, or two Z and the carbon atoms to which they are attached form a carbonyl group;
  • X is -H or -Cl
  • Y is -H or methyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-C6 alkyl
  • X is -H or -Cl
  • Y is -H or methyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-C6 alkyl
  • n is selected from any integer from 1-20. m is preferably 1-10, more preferably 5-10.
  • the main steps of preparing anti-HER2-antibody drug conjugates include:
  • the anti-HER2 antibody After diluting the anti-HER2 antibody, it is incompletely reduced with dithiothreitol (DTT). After incubation, it is eluted and exchanged with Sephadex G 25 resin to determine the number of sulfhydryl groups of the antibody.
  • Add the drug of formula V, VI, VII or VIII to the reduced antibody stir at room temperature, add cysteine and continue to stir to obtain; the drug and antibody of formula V, VI, VII or VIII
  • the molar equivalent ratio of the number of sulfhydryl groups is (1 to 2):1, and in some embodiments, the molar equivalent ratio of the drug described in formula V, VI, VII or VIII to the number of sulfhydryl groups of the antibody is 1.5:1.
  • the diluted anti-HER2 antibody is diluted to 7-9 mg/mL.
  • reaction mixture after the above-mentioned reaction mixture is ultrafiltered, it is purified with a gel filtration column equilibrated with G25 in a phosphate buffer with a pH value of 7.4 ⁇ 0.1.
  • the prepared anti-HER2-antibody drug conjugate is filtered with a 0.22 micron filter and stored at -80 ⁇ 5°C.
  • DTNB 5,5'dithiobis(2-nitrobenzoic acid)
  • DAR drug-antibody coupling ratio
  • the DAR in the anti-HER2-antibody drug conjugate prepared by the method described above is 2-8; in some embodiments, the DAR is 3-5; in some embodiments, the DAR is 3.3-3.7; In some embodiments, the DAR is about 3.5.
  • the concentration of the drug of formula V, VI, VII or VIII can be measured by ultraviolet absorption, the aggregation rate can be measured by size exclusion chromatography, and the residual free drug can be measured by reversed-phase high performance liquid chromatography.
  • the main steps of preparing the anti-HER2-antibody drug conjugate include: diluting the anti-HER2 antibody with solution B (50mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 8.0) to 8.0mg/mL, Then incomplete reduction with DTT (6 molar equivalents). After incubating for 60 minutes at 37°C, it was exchanged with solution A (50mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 6.5) with Sephadex G 25 resin.
  • solution B 50mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 8.0
  • DTT 6 molar equivalents
  • the sulfhydryl antibody value is determined by measuring the absorbance, and the sulfhydryl concentration is determined by the reaction product of the sulfhydryl and DTNB, and then the absorbance at 412nm is measured.
  • the concentration of diethylformamide (DMA) during the coupling reaction is 10% (V/V).
  • the ratio of the number of sulfhydryl groups of the drug of formula V, VI, VII or VIII to the anti-HER2 antibody is 1.5:1.0 (molar equivalent).
  • Add the drugs of formula V, VI, VII or VIII to the reduced anti-HER2 antibody stir at room temperature for 3 hours, add 5 mM cysteine and continue stirring for 1 hour to obtain.
  • the anti-HER2-ADC represented by formula IV is ADC1, wherein the anti-HER2 antibody Abu is antibody A, the light chain sequence of antibody A is SEQ ID NO.1, and the heavy chain sequence of antibody A is SEQ ID NO. .2.
  • the anti-HER2-ADC represented by formula IV is ADC2, wherein the anti-HER2 antibody Abu is antibody B, the light chain sequence of antibody B is SEQ ID NO. 3, and the heavy chain sequence of antibody A is SEQ ID NO. .4.
  • the anti-HER2-ADC represented by formula IV is ADC3, wherein the anti-HER2 antibody Abu is trastuzumab.
  • the anti-HER2-ADC represented by Formula IV is ADC4, wherein the anti-HER2 antibody Abu is Pertuzumab.
  • the anti-PD-1 antibody is the anti-PD-1 antibody disclosed in US Pat. No. 8,354,509.
  • the anti-PD-1 antibody is Antibody C, or Nivolumab (Nivolumab), or Pembrolizumab (Pembrolizumab), or Tereprizumab (JS001), or Sindili Monoclonal antibody (Sintilimab), or Tislelizumab (Tislelizumab), or Carrelizumab (Camrelizumab), or Genolimzumab (Genolimzumab), or any of two or more of the above anti-PD-1 antibodies combination.
  • the anti-PD-1 antibody is antibody C
  • the light chain sequence of antibody C is SEQ ID NO.5
  • the heavy chain sequence is SEQ ID NO.6.
  • Antibody A, Antibody B, and Antibody C are antibodies whose sequences are shown in Table 1, or whose amino acid sequence is shown in Table 1, and have one or more amino acid substitutions, but the biological activity is not changed.
  • the binding number of small molecule drugs corresponding to one anti-HER2 antibody in the anti-HER2-antibody drug conjugate antibody-drug conjugate that is, the drug binding number of the antibody, or called drug antibody Coupling ratio (DAR), selected from an integer between 1 and 10, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; when considering the average number of small molecule drugs combined , Which is the average drug binding number of the antibody, or the average drug-antibody coupling ratio (average DAR), and its value is the value of p, which is selected from a natural number between 1 and 10.
  • DAR drug antibody Coupling ratio
  • p is selected from 2-8; in some embodiments, p is selected from 3-5; in some embodiments, p is selected from 3.3-3.7; in some embodiments, p is selected from 3.5.
  • the weight ratio of the anti-PD-1 antibody and the anti-HER2-ADC is (1:10)-(10:1). For example, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1 5:1, 6:1, 7:1, 8:1, or 9:1, or the range between any two ratios, including the end point.
  • the weight ratio of the anti-PD-1 antibody to the anti-HER2-ADC is 1:1-1:10. For example, 1:9.5, 1:8.5, 1:7.5, 1:6.5, 1:5.5, 1:4.5, 1:3.5, 1:2.5, or 1:1.5, or the range between any two ratios, including end.
  • the weight ratio of the anti-PD-1 antibody to the anti-HER2-ADC is 1:1-1:2.
  • 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, or 1:1.9 or the range between any two ratios, including the end point.
  • the anti-PD-1 antibody and the anti-HER2-ADC can either be independent administration units or a combined unit for co-administration.
  • the anti-PD-1 antibody and the anti-HER2-ADC in the drug combination can be administered simultaneously, separately or sequentially.
  • the invention also provides an application of the combined medicine in the preparation of a kit, a medicine box, a medicine pack or a medicine for preventing and treating tumors.
  • the kit, kit, medicine pack or medicine contains the anti-PD-1 antibody and the independent dosing unit formed by the anti-HER2-ADC.
  • each independent administration unit is used in combination to prevent or treat tumors.
  • each independent administration unit may be administered simultaneously, sequentially or separately.
  • the kit, kit, kit or medicine also includes instructions for instructing the anti-tumor combination drug.
  • the tumor is a benign tumor or a malignant tumor.
  • malignant tumors are cancer.
  • the tumor includes, but is not limited to: Hodgkin’s lymphoma, non-Hodgkin’s lymphoma, prelymphoblastic lymphoma, small non-cleaved cell lymphoma, Burkitt’s lymphoma, non-primordial lymphoma Special lymphoma, diffuse large B cell lymphoma, anaplastic large cell lymphoma, kidney tumor, Wilms tumor, Wilms tumor, renal clear cell carcinoma, renal rhabdoid tumor, renal clear cell sarcoma, renal primitive neuroectodermal Leaf tumor, neuroblastoma, ganglion cell neuroblastoma, ganglion cell neuroma, extracranial germ cell tumor, mature teratoma, immature teratoma, endodermal sinus tumor, yolk sac tumor, seminoma , Dysgerminoma, choriocarcinoma, embryonic carcinoma, osteosarcoma, chondrosarcoma, rhabdomyos
  • the cancer includes, but is not limited to: nasopharyngeal cancer, papillary thyroid cancer, bowel cancer, breast cancer, stomach cancer, liver cancer, prostate cancer, breast cancer, lung cancer, cervical cancer, ovarian cancer, kidney cancer, lymphoma, leukemia, skin cancer Wait.
  • the tumor is a solid tumor.
  • the tumor is a HER2-positive solid tumor.
  • the tumor is a tumor with high expression of HER2, or a tumor with low expression of HER2.
  • the solid tumor is: HER2-positive melanoma, HER2-positive breast cancer, or HER2-positive gastric cancer.
  • the present invention also provides an application of the anti-tumor combined drug as described above in the preparation of a kit, a kit, a drug pack or a compound drug for inhibiting tumor cell proliferation.
  • the kit, the kit, the drug pack or the drug contains the anti-PD-1 antibody and the anti-HER2-antibody drug conjugate as the compound represented by formula I or a pharmaceutically acceptable salt thereof
  • each independent administration unit is used in combination to suppress the proliferation of tumor cells.
  • the independent administration units can be administered simultaneously, sequentially or separately.
  • the kit, kit, kit or medicine also includes instructions for instructing the anti-tumor combination drug.
  • the tumor cell is a tumor cell expressing HER2.
  • tumor cells expressing HER2 are tumor cells with high expression of HER2, or tumor cells with low expression of HER2.
  • the tumor cells with high HER2 expression are: breast cancer cells or gastric cancer cells; the tumor cells with low HER2 expression are: melanoma cells.
  • the tumor cell with high HER2 expression is: breast cancer cell SK-BR-3 or gastric cancer cell NCI-N87; the tumor cell with low expression of HER2 is: melanoma cell B16F10-hHER2.
  • Each dosing unit in the combined medicine of the present invention is separately prepared with an acceptable carrier in the medicine to obtain a pharmaceutical preparation.
  • the dosage form of the pharmaceutical preparation is a liquid preparation or a solid preparation.
  • the dosage form of the pharmaceutical preparation is water injection or lyophilized powder injection. If the dosage form of the pharmaceutical preparation is a water injection injection, when used, it can be diluted with a pharmaceutically acceptable solvent (such as water for injection) to the required concentration; if the dosage form of the pharmaceutical preparation is a freeze-dried powder injection, the pharmaceutical preparation can be used. The accepted solvent (such as water for injection) is dissolved, and then diluted according to the required concentration.
  • the present invention also provides a use of the anti-HER2-antibody drug conjugate in the preparation of an anti-tumor drug, which is used to treat patients who are receiving anti-PD-1 antibodies.
  • the present invention also provides a use of an anti-PD-1 antibody in the preparation of an anti-tumor drug, which is used to treat patients who are receiving anti-HER2-antibody drug conjugates.
  • a method for treating or inhibiting tumors of the present invention includes: administering an effective dose of anti-PD-1 antibody and anti-HER2-ADC to the patient.
  • a method for treating or inhibiting tumors of the present invention includes: administering an effective dose of anti-PD-1 antibody to a patient who is receiving anti-HER2-ADC treatment.
  • the method for treating or inhibiting tumors of the present invention includes: administering an effective dose of anti-HER2-ADC to a patient who is being treated with an anti-PD-1 antibody.
  • the anti-HER2-ADC is a compound represented by formula I or a pharmaceutically acceptable salt thereof;
  • Abu is an anti-HER2 antibody
  • p is selected from 1-10; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • X is -H or a halogen group
  • R 2 is -H or C1-C6 alkyl
  • R 4 is -OH or -SH
  • R 5 is C1-C6 alkyl or benzyl
  • R 6 is C1-C6 alkyl, phenyl or benzyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-6 alkyl
  • Z is independently -H or C1-C4 hydrocarbon group, or two Z and the carbon atoms to which they are attached form a carbonyl group;
  • the anti-HER2-ADC is:
  • Abu is an anti-HER2 antibody
  • p is selected from 1-10; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • X is -H or -Cl
  • Y is -H or methyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-C6 alkyl
  • the anti-HER2-ADC is:
  • Abu is an anti-HER2 antibody
  • p is selected from 1-10; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
  • X is -H or -Cl
  • Y is -H or methyl
  • R 7 is -H, C1-C6 alkyl or amino acid side chain
  • R 8 is -H or C1-C6 alkyl
  • n is selected from any integer from 1-20. m is preferably 1-10, more preferably 5-10.
  • the anti-HER2-ADC is:
  • Abu is an anti-HER2 antibody
  • p is selected from 1-10.
  • 1-10 For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • p When p represents an average DAR, p may be a non-integer, such as about 3.3 to about 3.7, or about 3.5.
  • the anti-PD-1 antibody and the anti-HER2-ADC are separate dosing units, and are used in combination; or the anti-PD-1 antibody and the anti-HER2-ADC simultaneously form a combined dosing unit , Combination medication.
  • the anti-PD-1 antibody and the anti-HER2-ADC are administered simultaneously, separately or sequentially.
  • the anti-PD-1 antibody may be administered before the anti-HER2-ADC, or after the anti-HER2-ADC, or at the same time as the anti-HER2-ADC.
  • the anti-HER2-ADC and anti-PD-1 antibodies are preferably separate administration units, and are administered to patients with HER2-positive cancer.
  • the patient when the anti-HER2-ADC and anti-PD-1 antibodies are combined together to form a combined dosing unit, the patient can be given an effective dose of anti-HER2-ADC and anti-PD-1 at the same time.
  • PD-1 antibody when the anti-HER2-ADC and anti-PD-1 antibodies are combined together to form a combined dosing unit, the patient can be given an effective dose of anti-HER2-ADC and anti-PD-1 at the same time.
  • a therapeutically effective amount of anti-HER2-ADC and anti-PD-1 antibodies are applied to the test patient separately or simultaneously.
  • the administration period of anti-HER2-ADC and anti-PD-1 antibody can be the same or different.
  • the dose of anti-HER2-ADC is generally 1mg/kg-10mg/kg.
  • the dose of anti-PD-1 antibody is generally 1mg/kg-10mg/kg, or 60mg-600mg per patient.
  • the unit dose of anti-HER2-ADC is about 1.2 mg/kg-6.0 mg/kg; in some embodiments, the unit dose of anti-HER2-ADC is about 1.2 mg/kg, or 2.4 mg/kg , Or 3.6 mg/kg, or 4.8 mg/kg, or 6.0 mg/kg; in some embodiments, the unit dose of anti-HER2-ADC is about 4.8 mg/kg. In some embodiments, the unit dose of anti-HER2-ADC is about 3.6 mg/kg.
  • the anti-HER2-ADC administration period is 1-28 days. In some embodiments, the anti-HER2-ADC administration cycle is 1-14 days, such as 2-4 days, or 3.5 days, that is, every 1-14 days, such as every 2-4 days, or every 3.5 days, respectively.
  • the patient has a unit dose of anti-HER2-ADC.
  • the anti-HER2-ADC administration period is 14-28 days, such as 20-22 days, or 21 days, that is, every 14-28 days, such as every 20-22 days, or every 21 days, respectively.
  • the patient has a unit dose of anti-HER2-ADC.
  • the unit dose of anti-PD-1 antibody is about 100 mg-200 mg. In some embodiments, the unit dose of anti-PD-1 antibody is about 100 mg, 150 mg, or 200 mg. In some embodiments, the unit dose of anti-PD-1 antibody is about 1 mg/kg-2 mg/kg to 200 mg; in some embodiments, the unit dose of anti-PD-1 antibody is about 2 mg/kg to 200 mg.
  • the anti-PD-1 antibody administration period is 1-49 days. In some embodiments, the anti-PD-1 antibody administration cycle is 1-14 days, such as 6-8 days, or 7 days, that is, every 1-14 days, such as every 6-8 days, or every 7 days, Give the patient a unit dose of anti-PD-1 antibody.
  • the anti-PD-1 antibody administration cycle is 14-28 days, such as 20-22 days, or 21 days, namely every 14-28 days, such as every 20-22 days, or every 21 days, Give the patient a unit dose of anti-PD-1 antibody.
  • the anti-PD-1 antibody administration cycle is 28-49 days, such as 35-49 days, or 41-43 days, or 42 days, that is, every 28-49 days, such as every 35-49 days. , Or every 41-43 days, or every 42 days, give the patient a unit dose of anti-PD-1 antibody.
  • 1.2 mg/kg-4.8 mg/kg or 2.4 mg/kg-3.6 mg/kg of anti-HER2-ADC and 100 mg-200 mg of anti-PD-1 antibody are administered to the patient every 21 days.
  • 1.2 mg/kg-4.8 mg/kg or 2.4 mg/kg-3.6 mg/kg of anti-HER2-ADC and 200 mg of anti-PD-1 antibody are administered to the patient every 21 days.
  • 1.2 mg/kg-4.8 mg/kg or 2.4 mg/kg-3.6 mg/kg of anti-HER2-ADC is applied to the patient every 21 days, and 100 mg-200 mg is applied to the patient every 42 days Anti-PD-1 antibody.
  • 1.2 mg/kg-4.8 mg/kg or 2.4 mg/kg-3.6 mg/kg of anti-HER2-ADC is applied to the patient every 21 days, and 200 mg of anti-PD is applied to the patient every 42 days -1 antibody.
  • the anti-HER2-ADC and anti-PD-1 antibody are administered separately or simultaneously as intravenous infusion.
  • the present invention When using anti-HER2-ADC to treat patients suffering from HER2-positive cancer, the present invention adds anti-PD-1 antibody to improve the therapeutic effect.
  • the efficacy of the anti-HER2-ADC of the present invention is positively correlated with the expression level of HER2, but no matter whether the expression level of HER2 in HER2-positive cancers is high or not, anti-PD-1 antibody is added, Both can improve the therapeutic effect or reduce the effective dose of anti-HER2-ADC.
  • the combination drug provided by the present invention can treat them and broaden the application range of anti-HER2-ADC.
  • the tumor treated by the combination of the present invention is a HER2-positive solid tumor.
  • the solid tumor is: HER2-positive melanoma, HER2-positive breast cancer, or HER2-positive gastric cancer.
  • the anti-PD-1 antibody and anti-HER2-ADC play a role in inhibiting tumor cell proliferation and/or anti-tumor in cooperation with peripheral blood mononuclear cells (PBMC) or T cells of the patient. Further, the anti-PD-1 antibody and anti-HER2-ADC are used in combination in a weight ratio of (1:10)-(10:1).
  • the anti-PD-1 antibody and anti-HER2-ADC are used in combination in a weight ratio of 1:1-1:10.
  • the anti-PD-1 antibody and anti-HER2-ADC are used in combination with a weight ratio of 1:1-1:2.
  • the anti-HER2-ADC is a compound of formula IV used in combination with an anti-PD-1 antibody (such as antibody C):
  • Abu is an anti-HER2 antibody
  • p is selected from 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the mean value of p is 3.3-3.7.
  • the light chain sequence of Abu is SEQ ID NO. 1, and the heavy chain sequence is SEQ ID NO. 2; or the light chain sequence of Abu is SEQ ID NO. 3 and the heavy chain sequence is SEQ ID NO. 4 .
  • the anti-HER2-ADC of formula IV is ADC1, wherein the light chain sequence of Abu is SEQ ID NO. 1, and the heavy chain sequence is SEQ ID NO. 2; the average value of p is about 3.5.
  • the anti-HER2-ADC of formula IV is ADC2, wherein the light chain sequence of Abu is SEQ ID NO. 3, and the heavy chain sequence is SEQ ID NO. 4; the average value of p is about 3.5.
  • the anti-PD-1 antibody is antibody C, or nivolumab, or pembrolizumab, or teriprizumab, or sintilizumab, or tislelizumab Anti-, or Carrelizumab, or Generosumumab, or any combination of the above PD-1 antibodies.
  • the anti-PD-1 antibody is antibody C
  • the light chain sequence of the antibody C is SEQ ID NO.5
  • the heavy chain sequence is SEQ ID NO.6.
  • the unit dose of ADC1 is about 1.2 mg/kg-6.0 mg/kg; in some embodiments, the unit dose of ADC1 is about 1.2 mg/kg, or 2.4 mg/kg, or 3.6 mg/kg , Or 4.8 mg/kg, or 6.0 mg/kg; in some embodiments, the unit dose of ADC1 is about 4.8 mg/kg. In some embodiments, the unit dose of ADC1 is about 3.6 mg/kg.
  • the ADC1 administration cycle is 14-28 days, such as 20-22 days, or 21 days, that is, every 14-28 days, such as every 20-22 days, or every 21 days, ADC1 is administered to the patient once Unit dose.
  • the unit dose of Antibody C is about 100 mg-200 mg. In some embodiments, the unit dose of antibody C is about 100 mg, 150 mg, or 200 mg. In some embodiments, the unit dose of antibody C is about 1 mg/kg-2 mg/kg to 200 mg; in some embodiments, the unit dose of antibody C is about 2 mg/kg to 200 mg.
  • the antibody C administration cycle is 14-49 days. In some embodiments, the antibody C administration cycle is 14-28 days, such as 20-22 days, or 21 days, that is, every 14-28 days, such as every 20-22 days, or every 21 days, is administered to the patient once Antibody C unit dose. In some embodiments, the antibody C administration cycle is 28-49 days, such as 35-49 days, every 41-43 days, or 42 days, and the patient is given a unit dose of antibody C once.
  • ADC1 at 1.2 mg/kg to 4.8 mg/kg or 2.4 mg/kg to 3.6 mg/kg and 100 mg to 200 mg of antibody C are administered to the patient every 21 days.
  • 1.2 mg/kg-4.8 mg/kg or 2.4 mg/kg-3.6 mg/kg of ADC1 and 200 mg of antibody C are administered to the patient every 21 days.
  • ADC1 at 1.2 mg/kg to 4.8 mg/kg or 2.4 mg/kg to 3.6 mg/kg is applied to the patient every 21 days, and antibody C at 100 mg to 200 mg is applied every 42 days.
  • ADC1 at 1.2 mg/kg to 4.8 mg/kg or 2.4 mg/kg to 3.6 mg/kg is applied to the patient every 21 days, and antibody C at 200 mg is applied every 42 days.
  • ADC1 and antibody C are administered separately or simultaneously as intravenous infusion.
  • Staphylococcal enterotoxin B SEB: Academy of Military Medical Sciences SL008.
  • SK-BR-3 breast cancer cells
  • NCI-N87 gastric cancer cells
  • B16F10/hHER2 melanoma cells
  • PBMC or T cell Reid Biotechnology Co., Ltd.
  • ADC1 is serially diluted, starting at 10 nM, and then diluted 3 times, a total of 9 gradients, the concentration of the 10th well is 0.
  • the microplate reader reads the plate at an absorbance of 450.
  • T cells are mixed with SK-BR-3 cells 10:1, and SK-BR-3 cells are 1 ⁇ 10 4 /well.
  • the results are shown in Figure 1.
  • the antibody C+ADC1 combination group had significantly lower readings than the ADC1 single administration group.
  • the antibody C+ADC1 combination group had ADC1 IC50 value of 0.055nM.
  • the IC50 value of ADC1 was 0.065nM. It can be seen that in the presence of T cells, the addition of antibody C enhances the inhibitory effect of ADC1 on SK-BR-3 as a whole.
  • T cells and NCI-87 cells are mixed at 10:1
  • the results are shown in Figure 2.
  • the IC50 value of the antibody C+ADC1 combination group was significantly lower than that of the ADC1 single administration group. Among them, in the antibody C+ADC1 combination group, the IC50 value of ADC1 was 0.075nM. In the ADC1 single administration group, the IC50 value of ADC1 was 0.261 nM. It can be seen that in the presence of T cells, the addition of antibody C enhances the inhibitory effect of ADC1 on NCI-87 as a whole.
  • Reagents FACS buffer; 3% FBS (Grnco CAT#100991-148); 2mM EDTA (Grnco CAT#15575-038); SterI Ca 2+ and Mg 2+ PBS (1X, Stock); Mouse Fc blocking reagent: Purified Rat Anti-Mouse CDI6/CD32 (Mouse BD Fc Block TM CAT#553141); Human Fc blocking reagent: FcR Blocking Reagent, human (Miltenyi Biotec CAT#130-059-901); PBS (Hyclone CAT#SH0256.01); UltraComp eBeads (eBioscience CAT#01-2222-42).
  • FACS antibody hCD340 (hHER2), fluorescein APC, clone number 24D2, homosexual control Mouse IgG1, ⁇ , manufacturer Biolegend. 7-AAD, manufacturer BD.
  • the electronic data generated by the flow cytometer is analyzed with Kaluza software.
  • the IC50 of ADC1 was determined in the B16F10/hHER2 cell line (9 concentrations, 3 replicates). The 9 concentration gradients are 240nM, 48nM, 9.6nM, 1.92nM, 0.384nM, 76.8pM, 15.36pM, 3.07pM, 0.61pM, respectively. Cisplatin served as a positive control.
  • the stock solution concentration of the ADC drug to be tested is 20 mg/ml, which is 133330 nM. Perform 5-fold serial dilutions with medium.
  • Figure 6 shows the IC50 curve of ADC1 in B16F10-hHER2 cells, and the calculated IC50 value of ADC1 is 61 nM.
  • Figure 7 shows the IC50 curve of the positive control Cisplatin in B16F10-hHER2 cells. The calculated IC50 value of Cisplatin is 38.7 ⁇ M.
  • hPD1/hPDL1 KI HuGEMM mice were subcutaneously inoculated with B16F10-hHER2 cells to establish a subcutaneous transplantation tumor model of melanoma.
  • the test is divided into test drug ADC1 high dose (10mg/kg, QW), medium dose (3mg/kg, QW) and low dose (1mg/kg, QW), test drug antibody C (10mg/kg, BIW), and test The drug ADC1 high, medium and low doses were administered in combination with the test drug antibody C.
  • 6 rats in each group were injected into the tail vein for two weeks.
  • the efficacy is evaluated based on the relative tumor inhibition rate (TOI), and the safety is evaluated based on the changes in animal weight and death.
  • QW refers to the dosing cycle once a week; BIW refers to the dosing cycle twice a week.
  • the route of administration is i.v (tail vein injection).
  • the grouping day is day0, and the administration day is day0.
  • mice HPD1/hPDL1 K1 HuGEMM mice, female, 6-8 weeks (the age of mice at the time of tumor cell inoculation), 83 mice were used for transfer. 48 mice entered the group, and the mice weighed 19-26.5g. Purchased from Shanghai Southern Model Biotechnology Co., Ltd. Feeding environment: SPF level.
  • Test product ADC1 white and slightly yellow freeze-dried powder injection, packaging specification: 100mg/bottle, 1 bottle in total, sealed and stored at 4°C.
  • Test product antibody C clear or milky white injection, packaging specification: 2mg/ml/bottle, 1 bottle in total, 4ml in total, sealed and stored at 4°C.
  • B16F10-hHER2 cells were cultured in DMEM medium containing 10% fetal bovine serum and 0.5 ⁇ g/ml Puromycin. B16F10-hHER2 cells in the exponential growth phase were collected and resuspended in PBS to a suitable concentration for subcutaneous tumor inoculation in mice.
  • the collected B16F10-hHER2 (1 ⁇ 10 5 ) cells in the exponential growth phase were resuspended in 0.1 ml of PBS (0.1 ml/mouse) and inoculated subcutaneously on the right back of experimental mice, and tumor growth was regularly observed.
  • mice When the average tumor volume of the tumor-bearing mice reached about 75 mm 3 , the mice were randomly grouped according to Table 3. The day of grouping is set to Day 0, and the administration starts on Day 0 (the tumor volume is measured on the day of grouping, and the tumor volume is not repeated during administration). The detailed method of administration, dosage and route of administration are shown in Table 3.
  • test substance, reference substance and positive reference substance solution is carried out in a biological safety cabinet or ultra-clean workbench.
  • the final concentration is 20mg/ml. Avoid shaking and vortexing, store the mother liquor at 2-8°C.
  • the second step of dilution According to the mother liquor reagent concentration of 20mg/ml, the solution diluted with water for injection should be stored at 2-8°C for no more than 4h.
  • the mother liquor According to the actual concentration of the mother liquor of 25mg/ml, use water for injection to dilute the corresponding multiple to obtain the administration concentration of 1mg/ml.
  • the diluted solution should be stored at 2-8°C for no more than 4h.
  • StudyDirector TM version number 3.1.399.19, supplier Studylog System, Inc., S. San Francisco, CA, USA
  • Relative tumor proliferation rate is the percentage value of the treatment group and the control group relative to the tumor volume or tumor weight at a certain point in time. Calculated as follows:
  • T/C% T RTV /C RTV ⁇ 100% (T RTV : average RTV of the treatment group; C RTV : average RTV of the vehicle control group;
  • V t is the tumor volume of the animal at the time of grouping
  • V t is the tumor volume of the animal after treatment
  • T/C% T TW /C TW ⁇ 100% (T TW : average tumor weight at the end of the experiment in the treatment group; C TW : average tumor weight at the end of the experiment in the vehicle control group)
  • TGI% (1-T/C) ⁇ 100%.
  • T and C are the relative tumor volume (RTV) or tumor weight (TW) at a specific time point in the treatment group and the control group, respectively).
  • the average tumor volume was 2719.61 ⁇ 508.94, and the treatment group tested ADC1 low-dose group (G02 1mg/kg) and medium-dose group (G03 3mg/kg)
  • the average tumor volume of the high-dose group (G04 10mg/kg) was 1784.89 ⁇ 349.96 (low-dose group), 2355.02 ⁇ 345.36 (medium-dose group), and 2335.15 ⁇ 276.99 (high-dose group), and none of them showed significant tumors. Inhibition (p>0.05).
  • Test drug ADC1 and test drug BAT13 06 combined administration group (G06 ADC1 (1mg/kg) + antibody C (10 mg/kg), G07 ADC1 (3 mg/kg) + antibody C (10 mg/kg), G08 ADC1 (10 mg/kg) kg)+antibody C (10mg/kg)), G08 (ADC1(10mg/kg)+IBATI306(10mg/kg)) has an average tumor volume of 1292.59 ⁇ 314.22 and a TGI of 49.62%, showing a significant tumor suppressor effect (p ⁇ 0.05).
  • the combined administration group G08 (ADC1 (10 mg/kg) + BAT 1306 (10 mg/kg)) showed a significant tumor suppressor effect compared to the ADC1 high-dose group G04 (10 mg/kg) (p ⁇ 0.05).
  • the expression level of HER2 in tumor models is positively correlated with the efficacy of ADC1. Because the expression level of HER2 of B16F10/hHER2 is not high, the efficacy of ADC1 in vivo and in vitro is not obvious. But after combined with antibody C, it showed a significant tumor suppressing effect.
  • Dosage records (shown by the arrow in Figure 8): day0 all groups; day3 (antibody C BIW): G01 (vehicle)/G05/G06/G07/G08; day7 all groups; day10 (antibody C BIW): G01 (vehicle) G05/G06/G07/G08.
  • the data is expressed as "mean value ⁇ standard error"; the rate of weight change is compared with the weight on the day of administration.
  • Test drug ADC1 high dose (10mg/kg, QW), medium dose (3mg/kg, QW) and low dose (1mg/kg, QW), test drug antibody C (10mg/kg, BIW), and test drug ADC1 high
  • the medium and low doses were combined with the test drug antibody C. No animals in each treatment group died due to obvious drug toxicity, and they were well tolerated during the treatment. The combined application of ADC1 and antibody C did not cause adverse reactions in mice , Drug safety.

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Abstract

一种抗肿瘤的联合用药物，包括抗HER2-抗体药物偶联物和抗PD-1抗体；所述抗HER2-抗体药物偶联物与所述抗PD-1抗体可以分别成为独立的给药单元，或所述抗HER2-抗体药物偶联物与所述抗PD-1抗体可以混合形成一个组合的给药单元。

Description

抗肿瘤的联合用药物及其应用 技术领域
本发明涉及医药生物学领域，特别是涉及一种抗肿瘤的联合用药物及其应用。
背景技术
肿瘤的发病率逐年上升，抗肿瘤药物的使用也逐渐增多。近年来，肿瘤治疗研究取得了重大进展，尤其是一些抗体药物在恶性肿瘤治疗中显示出了良好疗效。然而，只有少部分患者可达到长期生存。为了进一步提高药物在抗肿瘤治疗时的临床疗效，在使用抗肿瘤药物时，可采用联合用药。
联合用药需要合适的药物配伍，但如果联用的药物选择不当可能达不到理想的结果，甚至适得其反。主要原因是联合用药过程中，药物在体内发生复杂的反应，而并非简单的一加一等于二的作用。选择合适的药物联合用药，可使药效加强或副作用减轻。但药物选择不当，则可使药效减弱或出现不应有的毒副作用。有报道称，目前因药物相互作用引起的不良反应发生率超过30％。
因此，有必要提供一种抗肿瘤的联合用药物，实现药效的提高，同时保证用药安全。
发明内容
基于此，本发明的目的在于提供一种安全、高效的抗肿瘤的联合用药物。
为实现上述目的，本发明提供了如下技术方案：
一种抗肿瘤的联合用药物，包括抗HER2-抗体药物偶联物和抗PD-1抗体。在一些实施方式中，所述抗HER2-抗体药物偶联物与所述抗PD-1抗体分别成为独立的给药单元。在一些实施方式中，所述抗HER2-抗体药物偶联物与所述抗PD-1抗体共同形成组合的给药单元。
在一些实施方式中，所述抗HER2-抗体药物偶联物为如式Ⅰ所示的化合物或其药学上可接受的盐，
Figure PCTCN2020091753-appb-000001
其中，Abu为抗HER2抗体，p选自1-10；
X为-H或卤素基；
Y选自-H、C1-C6烷基、C3-C6环烷基或-C(＝O)R 5；
R 1选自-H、-OH、-OC(＝O)R 5或-OR 5基团；
R 2为-H或C1-C6烷基；
R 3为甲基﹑-CH 2OH或-CH 2OC(＝O)R 6；
R 4为-OH或-SH；
R 5为C1-C6烷基或苄基；
R 6为C1-C6烷基﹑苯基或苄基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-6烷基；
Z独立地为-H或C1-C4烃基，或两个Z与其所连接的碳原子形成一个羰基；
L选自任选被取代的C1-C20亚烃基或C3-C8环亚烃基，其中一个或多个-CH 2-基团独立地任选地被选自以下的基团所替代：C3-C8环亚烃基、-O-、-S-﹑-NR 8-﹑-C(＝O)-﹑-C(＝O)NR 8-﹑-NR 8C(＝O)-﹑-SO 2NR 8-或-NR 8SO 2-。
本发明还提供了一种如上所述的抗肿瘤的药物组合的应用。具体技术方案如下：
如上所述的抗肿瘤的联合用药物在制备抑制肿瘤细胞增殖的试剂盒、药盒、药包或药物中的应用。
如上所述的抗肿瘤的联合用药物在制备预防和治疗肿瘤的试剂盒、药盒、药包或药物中的应用。
一种治疗肿瘤的方法，包括：向需要治疗的患者给予有效剂量的抗PD-1抗体和抗HER2-抗体药物偶联物。
基于上述技术方案，本发明具有以下有益效果：
本发明发明人在研究中发现，将抗PD-1抗体和抗HER2-抗体药物偶联物联合用药时，抗PD-1抗体和抗HER2-抗体药物偶联物两者相互配合，可以发挥协同增效的作用，提高抑制肿瘤的效果。其中，由抗HER2抗体及与其连接的一个或多个化学药物形成的抗HER2-抗体药物偶联物，通过抗体依赖的细胞介导的细胞毒性作用和化学药物共同起效，抗HER2抗体还能实现对化学药物进行靶向投递，因此进一步显著增加联合用药物对肿瘤细胞的杀伤作用。同时，抗PD-1抗体作为免疫检查位点抑制剂，能够刺激细胞的肿瘤杀伤功能，从而显著提高了对肿瘤的抑制效果。而且本发明的联合用药物，在提高对肿瘤的抑制效果的同时，肿瘤患者还具有良好的耐受性，达到了安全、高效的用药目的。
附图说明
图1为T细胞存在时，抗体C和ADC1联合用药以及ADC1单独给药对于SK-BR-3细胞增殖的抑制效果曲线；
图2为T细胞存在时，抗体C和ADC1联合用药以及ADC1单独给药对于NCI-87细胞 增殖的抑制效果曲线；
图3为PBMC存在时，抗体C和ADC1联合用药以及ADC1单独给药对于SK-BR-3细胞增殖的抑制效果曲线；
图4为NCI-87细胞的FACS分析靶抗原(HER2)的表达结果；
图5为B16F10-hHER2细胞FACS分析靶抗原(HER2)的表达结果；
图6为ADC1对B16F10-hHER2细胞增殖的抑制效果曲线；
图7为阳性药Cisplatin对B16F10-hHER2细胞增殖的抑制效果曲线；
图8为抗体C和ADC1联合用药以及ADC1单独给药对于小鼠肿瘤体积的影响变化趋势图；
图9为抗体C和ADC1联合用药以及ADC1单独给药对于小鼠的体重影响变化趋势图。
具体实施方式
为了便于理解本发明，下面将参照实施例对本发明进行更全面的描述，以下给出了本发明的较佳实施例。但是，本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。应理解，下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用试剂，均为市售产品。
除非另有定义，本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本文所用术语“抗体”是指表现所需生物学活性(例如抑制配体与其受体的结合或通过抑制配体诱导的受体信号转导)的抗体的任何形式。因此，“抗体”以其最广泛的意义来施用，并明确包括但不限于单克隆抗体(包括全长单克隆抗体)、多克隆抗体和多特异性抗体。
本发明中术语“单抗”也称“单克隆抗体”，是指从基本上相同抗体群众获得的抗体，构成所述群的各个抗体是一致的。单克隆抗体具有高度特异性，可针对单个的抗原位点。此外，与通常包括针对多个不同决定簇(表位)的多种不同抗体的常规(多克隆)抗体制备物相反，每种单克隆抗体仅针对抗原上的单个决定簇。例如，本发明实施例中的表述抗HER2抗体(Anti-HER2 antibody)是指以原癌基因人类表皮生长因子受体2(human epidermal growth factor receptor-2，HER2)为靶标蛋白的抗体，抗PD-1抗体(Anti-PD-1 antibody)是指以程序性细胞死亡蛋白-1(programmed death-1，PD-1)为靶标蛋白的抗体。
本发明中术语“抗体-药物偶联物”或“ADC”是指与一个或多个化学药物(其可以任选地是治疗剂或细胞毒性剂)化学连接的结合蛋白如抗体或其抗原结合片段。在一些实施方案中，ADC包括抗体、细胞毒性或治疗药物和使得药物能够与抗体连接或偶联的接头。ADC通常 具有与抗体偶联的1、2、3、4、5、6、7、8、9或10个数的药物。可以包括在ADC中的药物有但不限于：有丝分裂抑制剂、抗肿瘤抗生素、免疫调节剂、基因治疗的载体、烷化剂、抗血管生成剂、抗代谢物、含硼试剂、化疗保护剂、激素、抗激素剂、皮质类固醇、光活性治疗剂、寡核苷酸、放射性核素试剂、拓扑异构酶抑制剂、酪氨酸激酶抑制剂和放射致敏剂。在一些实施方案中，包括在ADC中的药物可以是类美登素药物。在一些实施方案中，包括在ADC中的药物可以是如本申请所述的如式Ⅰ所示的化合物或其药学上可接受的盐。在一些实施方案中，在ADC中，抗体通过自身半胱氨酸或疏基化的氨基酸如疏基化赖氨酸，形成二硫键，与药物偶联。
本文所用术语“烷基”和“亚烷基”意指包括具有特定碳原子数目的支链的和直链的饱和脂肪烃基。例如，“C1-C6烷基”中“C1-C6”的定义包括以直链或支链排列的具有1、2、3、4、5或6个碳原子的基团。例如，“C1-C6”具体包括甲基、乙基、正丙基、异丙基、正丁基、叔丁基、异丁基、戊基(包括8种同分异构体)、己基(包括23种同分异构体)。术语“环烷基”指具有特定碳原子数目的单环饱和脂肪烃基。例如“环烷基”包括环丙基、甲基-环丙基、2,2-二甲基-环丁基、2-乙基-环戊基、环己基等。
本文中所用术语“卤素”包括氟、氯、溴和碘。
本文中所用术语“氨基酸侧链”指取代氨基酸中某一个基团(如氢原子)的取代基；如甘氨酸侧链，为取代甘氨酸亚甲基上一个氢原子形成的取代基。氨基酸侧链实例包括，但不限于天然氨基酸侧链。
本文所用术语“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。或者，“约”或“基本上包含”可意味着至多±20％的范围，比如±10％，±5％或±1％范围。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为该具体值的可接受误差范围内。
当用“给予”和“治疗”提及动物、人、实验对象、细胞、组织、器官或生物液时，是指将外源性药物、治疗剂、诊断剂或组合物与动物、人、受治疗者、细胞、组织、器官或生物液接触。“给予”和“治疗”可指例如治疗方法、药动学方法、诊断方法、研究方法和实验方法。治疗细胞包括让试剂与细胞接触以及让试剂与流液接触，其中所述流液与细胞接触。“给予”和“治疗”还意味着例如通过试剂、诊断剂、结合组合物或通过其他细胞对细胞进行体外和离体治疗。
本文所用“抑制”包括延缓与疾病有关的症状的发展和/或减轻所述疾病将要或预期发展的这些症状的严重程度。所述术语还包括减缓已有症状、防止另外的症状和减缓或防止这些症状的潜在原因。因此，所述术语表示业已将有益结果赋予患有疾病的脊椎动物对象。
本文所用术语“治疗有效量”或“有效量”是指当将抗HER2-抗体药物偶联物(抗HER2-ADC)和/或抗PD-1抗体或其片段单独或联合给予细胞、组织或受治疗者时，其有效 防止或减缓待治疗的疾病或病症的量。治疗有效剂量进一步指抗体药物偶联物(ADC)和/或抗体或其片段足以导致症状减缓的量，所述减缓症状例如为治疗、治愈、防止或减缓相关医学状态，或提高对所述病征的治疗率、治愈率、防止率或减缓率。对具体受治疗者的有效量可视多种因素而变化，例如待治疗的疾病、患者的整体健康状况、给药的方法途径和剂量及副作用的严重性。有效量可为避免显著副作用或毒性作用的最大剂量或给药方案。当施用给患者单独给予的活性成分时，治疗有效量是指该单独的成分。当施用组合时，治疗有效量是指产生治疗效果的活性成分的联合的量，而不论其是联合给予、连续给予还是同时给予。治疗有效量将减轻症状通常至少10％；通常至少20％；至少约30％；至少40％或至少50％。
抗HER2-抗体药物偶联物或抗PD-1抗体抗体可以形成广泛多种医药学上可接受的盐，包括但不限于：与有机酸形成的酸加成盐，这种有机酸包括但不限于脂肪族单羧酸和二羧酸、苯基取代的烷酸、羟基烷酸、烷二酸、芳香酸、脂肪族和芳香族磺酸、氨基酸等等，例如乙酸、三氟乙酸、丙酸、乙醇酸、丙酮酸、草酸、马来酸、丙二酸、琥珀酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸，等等；与无机酸反应形成的酸加成盐，这种无机酸包括盐酸、氢溴酸、硫酸、硝酸、磷酸、氢碘酸、氢氟酸、亚磷酸，等等；及与金属离子(例如，碱金属离子(例如钠或钾)、碱土金属离子(例如钙或镁)或铝离子)或者与有机碱如二乙醇胺、三乙醇胺、N-甲基葡糖胺等等形成的盐。本文中所述抗HER2-抗体药物偶联物或抗PD-1抗体抗体包括其医药学上可接受的盐。
如本文所用的术语“个体”、“受试者”或“患者”指的是作为治疗、观测或实验的目标的动物。仅举例来说，患者可以是(但不限于)哺乳动物，包括(但不限于)人类。
本发明提供一种抗肿瘤的联合用药物，包括：抗HER2-抗体药物偶联物(抗HER2-ADC)和抗PD-1抗体；。在一些实施方式中，抗HER2-抗体药物偶联物和抗PD-1抗体分别成为独立的给药单元。在一些实施方式中，，或抗HER2-抗体药物偶联物和抗PD-1抗体共同形成组合的给药单元。
在一些实施方式中，抗HER2-抗体药物偶联物和抗PD-1抗体分别成为独立的给药单元。这样一方面可以提高抗HER2-ADC和抗PD-1抗体在分别存放时的稳定性；另一方面，在实际使用过程中，能够灵活调整抗HER2-ADC和抗PD-1抗体的用法用量。
在一些实施方式中，在使用联合用药物时，抗HER2-ADC和抗PD-1抗体可以同时、依次或分别给药。抗PD-1抗体可以在施加抗HER2-ADC之前给药，和/或在施加抗HER2-ADC之后给药，和/或在施加抗HER2-ADC的同时给药。因此抗HER2-ADC和抗PD-1抗体优选分别成为独立的给药单元，便于施加在患有HER2阳性癌症的患者身上。联合给药，是指抗HER2-ADC和抗PD-1抗体在同一治疗周期进行给药以至于患者体内同时有有效剂量的抗HER2-ADC和抗PD-1抗体。；联合给药可以是抗HER2-ADC和抗PD-1抗体在同一天或不同天进行给药。实际操作允许时，也可以在同一时刻进行给药。
在一些实施方式中抗HER2-抗体药物偶联物为如式I所示的化合物或其药学上可接受的 盐，
Figure PCTCN2020091753-appb-000002
其中，Abu为抗HER2抗体，p选自1-10；
X为-H或卤素基；
Y选自-H、C1-C6烷基、C3-C6环烷基或-C(＝O)R 5；
R 1选自-H、-OH、-OC(＝O)R 5或-OR 5基团；
R 2为-H或C1-C6烷基；
R 3为甲基﹑-CH 2OH或-CH 2OC(＝O)R 6；
R 4为-OH或-SH；
R 5为C1-C6烷基或苄基；
R 6为C1-C6烷基﹑苯基或苄基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-6烷基；
Z独立地为-H或C1-C4烃基，或两个Z与其所连接的碳原子形成一个羰基；
L选自任选被取代的C1-C20亚烃基或C3-C8环亚烃基，其中一个或多个-CH 2-基团独立地任选地被选自以下的基团所替代：C3-C8环亚烃基、-O-、-S-﹑-NR 8-﹑-C(＝O)-﹑-C(＝O)NR 8-﹑-NR 8C(＝O)-﹑-SO 2NR 8-或-NR 8SO 2-。
优选地，L为-(CH 2) m-，其中m为整数，m＝1-20，优选为1-10，更优选为5-10。
在一些实施方式中，L中所述C1-C20亚烃基为被1到4个-SO 3H﹑-P(＝O)(OH) 2或R 23取代的C1-C20亚烃基，其中R 23各自独立地为任选地被独立地选自-SH﹑-S-C 1-4烷基﹑-CONR 11R 11以及-NR 11R 11的一个或两个基团取代的C 1-6烷基。
R 11选自-H、烷基、芳基、环烷基、环烯基、杂芳基和杂环，或者两个R 11和氮形成一个杂环，其中杂环中的碳可以选择用一个或两个氧取代。
在一些实施方式中，抗HER2-抗体药物偶联物为如式II所示的化合物或其药学上可接受的盐：
Figure PCTCN2020091753-appb-000003
其中，Abu为抗HER2抗体，p选自1-10；
X为-H或-Cl；
Y为-H或甲基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-C6烷基；
L选自任选被取代的C1-C20亚烃基或C3-C8环亚烃基，其中一个或多个-CH 2-基团独立地任选地被选自以下的基团所替代：C3-C8环亚烃基、-O-、-S-﹑-NR 8-﹑-C(O)-﹑-C(＝O)NR 8-﹑-NR 8C(＝O)-﹑-SO 2NR 8-或-NR 8SO 2-。
优选地，L为-(CH 2) m-，其中m为整数，m＝1-20，优选为1-10，更优选为5-10。
在一些实施方式中，L中所述C1-C20亚烃基为被1到4个-SO 3H﹑-P(＝O)(OH) 2或R 23取代的C1-C20亚烃基，其中R 23各自独立地为任选地被独立地选自-SH﹑-S-C 1-4烷基﹑-CONR 11R 11以及-NR 11R 11的一个或两个基团取代的C 1-6烷基。
在一些实施方式中，抗HER2-抗体药物偶联物为如式Ⅲ所示的化合物或其药学上可接受的盐：
Figure PCTCN2020091753-appb-000004
其中，Abu为抗HER2抗体，p选自1-10；
X为-H或-Cl；
Y为-H或甲基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-C6烷基；
m选自1-20中的任一整数。m优选为1-10，更优选为5-10。
在一些实施方式中，抗HER2-抗体药物偶联物为如式IV所示的化合物或其药学上可接受的盐：
Figure PCTCN2020091753-appb-000005
其中，Abu为抗HER2抗体，p选自1-10。
在一些实施方式中，所述抗HER2-抗体药物偶联物中的抗HER2抗体为抗体A、抗体B、曲妥珠单抗(Trastuzumab)和帕妥珠单抗(Pertuzumab)中的一种或多种；
其中，抗体A的轻链的氨基酸序列如SEQ ID NO.1所示，抗体A的重链的氨基酸序列如SEQ ID NO.2所示；或抗体A的轻链或重链序列的氨基酸有一个或多个取代，但生物活性不变。抗体B的轻链的氨基酸序列如SEQ ID NO.3所示，抗体B的重链的氨基酸序列如SEQ ID NO.4所示；或抗体B的轻链或重链序列的氨基酸有一个或多个取代，但生物活性不变。
在一些实施方式中，在抗HER2-抗体药物偶联物中，药物可以通过连接子偶联至抗HER2抗体Abu上。其中，所述药物和连接子连接后的结构选自如式V、VI、VII、或VIII所述的化合物：
Figure PCTCN2020091753-appb-000006
其中，X为-H或卤素基；
Y选自-H、C1-C6烷基、C3-C6环烷基或-C(＝O)R 5；
R 1选自-H、-OH、-OC(＝O)R 5或-OR 5基团；
R 2为-H或C1-C6烷基；
R 3为甲基﹑-CH 2OH或-CH 2OC(＝O)R 6；
R 4为-OH或-SH；
R 5为C1-C6烷基或苄基；
R 6为C1-C6烷基﹑苯基或苄基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-6烷基；
Z独立地为-H或C1-C4烃基，或两个Z与其所连接的碳原子形成一个羰基；
L选自任选被取代的C1-C20亚烃基或C3-C8环亚烃基，其中一个或多个-CH 2-基团独立地任选地被选自以下的基团所替代：C3-C8环亚烃基、-O-、-S-﹑-NR 8-﹑-C(＝O)-﹑-C(＝O)NR 8-﹑-NR 8C(＝O)-﹑-SO 2NR 8-或-NR 8SO 2-。
优选地，L为-(CH 2) m-，其中m为整数，m＝1-20，优选为1-10，更优选为5-10。
在一些实施方式中，L中所述C1-C20亚烃基为被1到4个-SO 3H﹑-P(＝O)(OH) 2或R 23取代的C1-C20亚烃基，其中R 23各自独立地为任选地被独立地选自-SH﹑-S-C 1-4烷基﹑-CONR 11R 11以及-NR 11R 11的一个或两个基团取代的C 1-6烷基。
Figure PCTCN2020091753-appb-000007
X为-H或-Cl；
Y为-H或甲基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-C6烷基；
L选自任选被取代的C1-C20亚烃基或C3-C8环亚烃基，其中一个或多个-CH 2-基团独立地任选地被选自以下的基团所替代：C3-C8环亚烃基、-O-、-S-﹑-NR 8-﹑-C(O)-﹑-C(＝O)NR 8-﹑-NR 8C(＝O)-﹑-SO 2NR 8-或-NR 8SO 2-。
优选地，L为-(CH 2) m-，其中m为整数，m＝1-20，优选为1-10，更优选为5-10。
在一些实施方式中，L中所述C1-C20亚烃基为被1到4个-SO 3H﹑-P(＝O)(OH) 2或R 23取代的C1-C20亚烃基，其中R 23各自独立地为任选地被独立地选自-SH﹑-S-C 1-4烷基﹑-CONR 11R 11以及-NR 11R 11的一个或两个基团取代的C 1-6烷基。
Figure PCTCN2020091753-appb-000008
X为-H或-Cl；
Y为-H或甲基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-C6烷基；
m选自1-20中的任一整数。m优选为1-10，更优选为5-10。
Figure PCTCN2020091753-appb-000009
在一些实施方式中，制备抗HER2-抗体药物偶联物的主要步骤包括：
稀释抗HER2抗体后，用二硫苏糖醇(DTT)不完全还原，孵育后经Sephadex G 25树脂洗脱交换，测定抗体的巯基数。将式V、VI、VII或VIII所述的药物加入已还原的抗体中，室温搅拌，加入半肮氨酸继续搅拌，即得；所述式V、VI、VII或VIII所述的药物与抗体巯基数的摩尔当量比为(1～2)：1，在其中一些实施方式中式V、VI、VII或VIII所述的药物与抗体巯基数的摩尔当量比为1.5:1。
在一些实施方式中，所述稀释抗HER2抗体为稀释至7～9mg/mL。
在一些实施方式中，上述反应的混合物经超滤后，用G25在pH值7.4±0.1的磷酸盐缓冲液平衡的凝胶过滤柱纯化。
在一些实施方式中，制备得到的抗HER2-抗体药物偶联物用0.22微米的滤器过滤，-80±5℃保存。
在一些实施方式中，用5,5’二硫代双(2硝基苯甲酸)(DTNB)测定式V、VI、VII或VIII所述的药物未反应的巯基数可得到药物/抗HER2抗体比率，即药物抗体偶联比(DAR)。
在一些实施方式中，如上所述的方法制备得到的抗HER2-抗体药物偶联物中的DAR为2-8；在一些实施方案中，DAR为3-5；在一些实施方案中，DAR为3.3-3.7；在一些实施方案中，DAR约为3.5。
在一些实施方式中，式V、VI、VII或VIII所述的药物可以通过紫外吸收测得浓度，通过尺寸排阻色谱测定聚集率，通过反向高效液相色谱法测定残留的游离药物。
在其中一个实施例中，制备抗HER2-抗体药物偶联物的主要步骤包括：将抗HER2抗体用溶液B(50mM磷酸钾，50mM NaCl和2mM EDTA，pH为8.0)稀释至8.0mg/mL，然后用DTT(6摩尔当量)不完全还原。在37℃孵育60分钟后，用溶液A(50mM磷酸钾，50mM NaCl和2mM EDTA，pH为6.5)经Sephadex G 25树脂洗脱交换。巯基抗体值通过测定吸 光度确定，通过巯基与DTNB的反应物，然后测定412nm处的吸收值来确定巯基的浓度。偶联反应时二乙基甲酰胺(DMA)的浓度为10％(V/V)。式V、VI、VII或VIII所述的药物与抗HER2抗体的巯基数的比率为1.5：1.0(摩尔当量)。将式V、VI、VII或VIII所述的药物加入己还原的抗HER2抗体中，室温搅拌3小时后，加入5mM半肮氨酸继续搅拌1小时，即得。
在一些实施方式中，式IV所示的抗HER2-ADC为ADC1，其中抗HER2抗体Abu为抗体A，抗体A的轻链序列为SEQ ID NO.1，抗体A的重链序列为SEQ ID NO.2。
在一些实施方式中，式IV所示的抗HER2-ADC为ADC2，其中抗HER2抗体Abu为抗体B，抗体B的轻链序列为SEQ ID NO.3，抗体A的重链序列为SEQ ID NO.4。
在一些实施方式中，式IV所示的抗HER2-ADC为ADC3，其中抗HER2抗体Abu为曲妥珠单抗。
在一些实施方式中，式IV所示的抗HER2-ADC为ADC4，其中抗HER2抗体Abu为帕妥珠单抗。
在一些实施方式中，抗PD-1抗体为美国专利US8,354,509中公开的抗PD-1抗体。
在一些实施方式中，所述抗PD-1抗体为抗体C，或纳武单抗(Nivolumab)，或派姆单抗(Pembrolizumab)，或特瑞普利单抗(JS001)，或信迪利单抗(Sintilimab)，或替雷利珠单抗(Tislelizumab)，或卡瑞利珠单抗(Camrelizumab)，或杰诺单抗(Genolimzumab)，或者上述抗PD-1抗体的两种以上的任意组合。
在一些实施方式中，所述抗PD-1抗体为抗体C，抗体C的轻链序列为SEQ ID NO.5，重链序列为SEQ ID NO.6。
抗体A、抗体B和抗体C的为序列如表1所示的抗体，或氨基酸序列如表1所示，且氨基酸有一个或多个取代，但生物活性不改变的抗体。
表1
Figure PCTCN2020091753-appb-000010
Figure PCTCN2020091753-appb-000011
在一些实施方式中，在所述抗HER2-抗体药物偶联物抗体-药物偶联物中一个抗HER2抗体对应的小分子药物的结合个数，即抗体的药物结合数，或称为药物抗体偶联比(DAR)，选自1至10之间的整数，或为1、2、3、4、5、6、7、8、9或10；当考虑小分子药物的平均结合个数时，即抗体的药物平均结合数，或称为平均药物抗体偶联比(平均DAR)，其值即为p的值，选自1至10之间的自然数。在一些实施方案中，p选自2-8；在一些实施方案中，p选自3-5；在一些实施方案中，p选自3.3-3.7；在一些实施方案中，p选自3.5。
在一些实施方式中，所述抗PD-1抗体和抗HER2-ADC的重量份数比为(1:10)-(10:1)。例如，1:9、1:8、1:7、1:6、1:5、1:4、1:3、1:2、1:1、2:1、3:1、4:1、5:1、6:1、7:1、8:1或9:1，或任何两个比例之间的范围，包括终点。
在一些实施方式中，所述抗PD-1抗体和和抗HER2-ADC的重量份数比为1:1-1:10。例如，1:9.5、1:8.5、1:7.5、1:6.5、1:5.5、1:4.5、1:3.5、1:2.5、或1:1.5，或任何两个比例之间的 范围，包括终点。
在一些实施方式中，所述抗PD-1抗体和和抗HER2-ADC的重量份数比为1:1-1:2。例如1:1.1、1:1.2、1:1.3、1:1.4、1:1.5、1:1.6、1:1.7、1:1.8或1:1.9，或任何两个比例之间的范围，包括终点。
本发明的联合用药物中，所述抗PD-1抗体与和抗HER2-ADC，既可以分别成为独立的给药单元，也可以形成共同给药的组合单元。当所述的药物组合分别成为独立的给药单元时，药物组合中所述抗PD-1抗体与所述和抗HER2-ADC在应用时，可以同时给药、分别或依次给药。
本发明还提供了一种所述联合用药物在制备预防和治疗肿瘤的试剂盒、药盒、药包或药物中的应用。
在一些实施方式中，所述试剂盒、药盒、药包或药物中，含有所述抗PD-1抗体和所述和抗HER2-ADC分别形成的独立的给药单元。优选地，各独立的给药单元联合用药，用于预防或治疗肿瘤。在一些实施方式中，各独立的给药单元可以同时、依次或分别给药。
在其中一些实施方式中，所述试剂盒、药盒、药包或药物中还包括有指导所述的抗肿瘤的联合用药物的说明书。
在一些实施方式中，肿瘤为良性肿瘤或恶性肿瘤。其中，恶性肿瘤即癌症。
在一些实施方式中，所述肿瘤包括但不限于：霍奇金淋巴瘤、非霍奇金淋巴瘤、前淋巴母细胞淋巴瘤、小无裂细胞淋巴瘤、伯基特淋巴瘤、非伯基特淋巴瘤、弥漫性大B细胞淋巴瘤、间变性大细胞淋巴瘤、肾脏肿瘤、肾母细胞瘤、Wilms瘤、肾透明细胞癌、肾横纹肌样瘤、肾透明细胞肉瘤、肾原始神经外胚叶瘤、神经母细胞瘤、节细胞神经母细胞瘤、节细胞神经瘤、颅外生殖细胞瘤、成熟畸胎瘤、未成熟畸胎瘤、内胚窦瘤、卵黄囊瘤、精原细胞瘤、无性细胞瘤、绒毛膜上皮癌、胚胎癌、骨肉瘤、软骨肉瘤、横纹肌肉瘤、软组织肉瘤、纤维肉瘤、恶性纤维组织细胞瘤、脂肪肉瘤、平滑肌肉瘤、血管肉瘤、淋巴管肉瘤、恶性神经鞘瘤、腺泡状软组织肉瘤、上皮样肉瘤、透明细胞肉瘤、恶性黑色素瘤、滑膜肉瘤、促纤维增生性小圆细胞瘤、尤文氏家族肉瘤：尤文氏肉瘤、原始神经外胚叶瘤、肝脏肿瘤、肝母细胞瘤、视网膜母细胞瘤、后颅窝髓母细胞瘤、胸腺瘤、肺母细胞瘤、胰母细胞瘤、胰岛细胞瘤、回盲部类癌、间皮瘤、黑色素瘤、间质细胞瘤、骨髓瘤或脑星形细胞瘤等。所述癌症包括但不限于：鼻咽癌、甲状腺乳头状癌、肠癌、乳癌、胃癌、肝癌、前列腺癌、乳腺癌、肺癌、宫颈癌、卵巢癌、肾癌、淋巴瘤、白血病、皮肤癌等。
在一些实施方式中，所述肿瘤为实体瘤。
在一些实施方式中，所述肿瘤为HER2阳性的实体瘤。
在一些实施方式中，所述肿瘤为HER2高表达的肿瘤，或HER2表达不高的肿瘤。
在一些实施方式中，所述实体瘤为：HER2阳性黑色素瘤、HER2阳性乳腺癌或HER2阳性胃癌。
本发明还提供了一种如上所述的抗肿瘤的联合用药物在制备抑制肿瘤细胞增殖的试剂盒、药盒、药包或复方药物中的应用。其中，所述试剂盒、药盒、药包或药物中，含有所述抗PD-1抗体和所述抗HER2-抗体药物偶联物如式Ⅰ所示的化合物或其药学上可接受的盐分别形成的独立的给药单元。优选地，各独立的给药单元联合用药，用于抑制肿瘤细胞的增殖。可选地，各独立的给药单元可以同时、依次或分别给药。
在其中一些实施方式中，所述试剂盒、药盒、药包或药物中还包括有指导所述的抗肿瘤的联合用药物的说明书。
在一些实施方式中，所述肿瘤细胞为表达HER2的肿瘤细胞。
其中，表达HER2的肿瘤细胞为HER2高表达的肿瘤细胞，或HER2表达不高的肿瘤细胞。
在一些实施方式中，所述HER2高表达肿瘤细胞为：乳腺癌细胞或胃癌细胞；所述HER2表达不高的肿瘤细胞为：黑色素瘤细胞。
在一些实施方式中，所述HER2高表达肿瘤细胞为：乳腺癌细胞SK-BR-3或胃癌细胞NCI-N87；所述HER2表达不高的肿瘤细胞为：黑色素瘤细胞B16F10-hHER2。
本发明所述的联合用药物中的各给药单元分别与药物中可接受的载体制备得到药物制剂。可选地，所述药物制剂的剂型为液体制剂或固体制剂。可选地，所述药物制剂的剂型为水针注射剂或冻干粉针剂。若药物制剂的剂型为水针注射剂时，使用时，可使用药学上可接受的溶剂(如注射用水)稀释至所需给药浓度；若药物制剂的剂型为冻干粉针剂，使用药学上可接受的溶剂(如注射用水)进行溶解，再根据所需的浓度进行稀释。
如上所述的抗肿瘤的联合用药物的应用，所述联合用药物用于在用抗HER2-抗体药物偶联物治疗患有HER2阳性癌症的患者过程中，同时、依次或分别施用给所述患者抗PD-1抗体。
本发明还提供了一种抗HER2-抗体药物偶联物用于制备抗肿瘤的药物中的用途，所述药物用于治疗正在接受抗PD-1抗体的患者。
本发明还提供了一种抗PD-1抗体用于制备抗肿瘤的药物中的用途，所述药物用于治疗正在接受抗HER2-抗体药物偶联物的患者。
本发明的一种治疗或抑制肿瘤的方法，包括：向患者给予有效剂量的抗PD-1抗体和抗HER2-ADC。
本发明的一种治疗或抑制肿瘤的方法，包括：向正在接受抗HER2-ADC治疗的患者给予有效剂量的抗PD-1抗体。
本发明的一种治疗或抑制肿瘤的方法，包括：向正在接受抗PD-1抗体治疗的患者给予有效剂量的抗HER2-ADC。
其中一些实施方式中，抗HER2-ADC为如式I所示的化合物或其药学上可接受的盐；
Figure PCTCN2020091753-appb-000012
其中，Abu为抗HER2抗体，p选自1-10；例如1、2、3、4、5、6、7、8、9或10；
X为-H或卤素基；
Y选自-H、C1-C6烷基、C3-C6环烷基或-C(＝O)R 5；
R 1选自-H、-OH、-OC(＝O)R 5或-OR 5基团；
R 2为-H或C1-C6烷基；
R 3为甲基﹑-CH 2OH或-CH 2OC(＝O)R 6；
R 4为-OH或-SH；
R 5为C1-C6烷基或苄基；
R 6为C1-C6烷基﹑苯基或苄基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-6烷基；
Z独立地为-H或C1-C4烃基，或两个Z与其所连接的碳原子形成一个羰基；
L选自任选被取代的C1-C20亚烃基或C3-C8环亚烃基，其中一个或多个-CH 2-基团独立地任选地被选自以下的基团所替代：C3-C8环亚烃基、-O-、-S-﹑-NR 8-﹑-C(＝O)-﹑-C(＝O)NR 8-﹑-NR 8C(＝O)-﹑-SO 2NR 8-或-NR 8SO 2-。
优选地，L为-(CH 2) m-，其中m为整数，m＝1-20，优选为1-10，更优选为5-10。
在一些实施方式中，L中所述C1-C20亚烃基为被1到4个-SO 3H﹑-P(＝O)(OH) 2或R 23取代的C1-C20亚烃基，其中R 23各自独立地为任选地被独立地选自-SH﹑-S-C 1-4烷基﹑-CONR 11R 11以及-NR 11R 11的一个或两个基团取代的C 1-6烷基。
在一些实施方式中，抗HER2-ADC为：
Figure PCTCN2020091753-appb-000013
其中，Abu为抗HER2抗体，p选自1-10；例如1、2、3、4、5、6、7、8、9或10；
X为-H或-Cl；
Y为-H或甲基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-C6烷基；
L选自任选被取代的C1-C20亚烃基或C3-C8环亚烃基，其中一个或多个-CH 2-基团独立地任选地被选自以下的基团所替代：C3-C8环亚烃基、-O-、-S-﹑-NR 8-﹑-C(O)-﹑-C(＝O)NR 8-﹑-NR 8C(＝O)-﹑-SO 2NR 8-或-NR 8SO 2-。
优选地，L为-(CH 2) m-，其中m为整数，m＝1-20，优选为1-10，更优选为5-10。
在一些实施方式中，L中所述C1-C20亚烃基为被1到4个-SO 3H﹑-P(＝O)(OH) 2或R 23取代的C1-C20亚烃基，其中R 23各自独立地为任选地被独立地选自-SH﹑-S-C 1-4烷基﹑-CONR 11R 11以及-NR 11R 11的一个或两个基团取代的C 1-6烷基。
在一些实施方式中，抗HER2-ADC为：
Figure PCTCN2020091753-appb-000014
其中，Abu为抗HER2抗体，p选自1-10；例如1、2、3、4、5、6、7、8、9或10；
X为-H或-Cl；
Y为-H或甲基；
R 7为-H、C1-C6烷基或氨基酸侧链；
R 8为-H或者C1-C6烷基；
m选自1-20中的任一整数。m优选为1-10，更优选为5-10。
在一些实施方式中，抗HER2-ADC为：
Figure PCTCN2020091753-appb-000015
其中，Abu为抗HER2抗体，p选自1-10。例如1、2、3、4、5、6、7、8、9或10。
当p表示平均DAR时，p可以是非整数，如约3.3-约3.7，或约3.5。
在一些实施方式中，所述抗PD-1抗体和所述抗HER2-ADC分别为独立的给药单元，联合用药；或所述抗PD-1抗体和抗HER2-ADC同时形成组合给药单元，联合用药。
在一些实施方式中，所述抗PD-1抗体和所述抗HER2-ADC同时、分别或依次给药。
在联合用药时，抗PD-1抗体可以在施加抗HER2-ADC之前给药，也可以在施加抗HER2-ADC之后给药，也可以在施加抗HER2-ADC的同时给药。抗HER2-ADC和抗PD-1抗体优选分别成为独立的给药单元，分别施加在患有HER2阳性癌症的患者身上。
或者，在其他一些实施方式中，联合用药时，抗HER2-ADC和抗PD-1抗体也可以混合在一起形成一个组合的给药单元后，向患者同时给予有效剂量的抗HER2-ADC和抗PD-1抗体。
在一些实施方式中，采用治疗有效量的抗HER2-ADC和抗PD-1抗体分别或者同时施加在受试患者上。抗HER2-ADC和抗PD-1抗体的给药周期可以相同或者不同。抗HER2-ADC剂量一般为1mg/kg-10mg/kg。抗PD-1抗体剂量一般为1mg/kg-10mg/kg，或者每个患者60mg-600mg。
在一些实施方式中，抗HER2-ADC的单位剂量约为1.2mg/kg-6.0mg/kg；在一些实施方式中，抗HER2-ADC的单位剂量约为1.2mg/kg，或2.4mg/kg，或3.6mg/kg，或4.8mg/kg，或6.0mg/kg；在一些实施方式中，抗HER2-ADC的单位剂量约为4.8mg/kg。在一些实施方式中，抗HER2-ADC的单位剂量约为3.6mg/kg。
在一些实施方式中，抗HER2-ADC给药周期为1-28天。在一些实施方式中，抗HER2-ADC给药周期为1-14天，比如2-4天，或3.5天，即分别每1-14天，比如每2-4天，或每3.5天，给予患者一次抗HER2-ADC单位剂量。
在一些实施方式中，抗HER2-ADC给药周期为14-28天，比如20-22天，或21天，即分别每14-28天，比如每20-22天，或每21天，给予患者一次抗HER2-ADC单位剂量。
在一些实施方式中，抗PD-1抗体单位剂量为约100mg-200mg。在一些实施方式中，抗PD-1抗体的单位剂量约为100mg，150mg，或200mg。在一些实施方式中，抗PD-1抗体的单位剂量约为1mg/kg-2mg/kg，至200mg；在一些实施方式中，抗PD-1抗体的单位剂量约为2mg/kg，至200mg。
在一些实施方式中，抗PD-1抗体给药周期为1-49天。在一些实施方式中，抗PD-1抗体给药周期为1-14天，比如6-8天，或7天，即分别每1-14天，比如每6-8天，或每7天，给予患者一次抗PD-1抗体单位剂量。
在一些实施方式中，抗PD-1抗体给药周期为14-28天，比如20-22天，或21天，即分别每14-28天，比如每20-22天，或每21天，给予患者一次抗PD-1抗体单位剂量。
在一些实施方式中，抗PD-1抗体给药周期为28-49天，比如35-49天，或41-43天，或 42天，即分别每28-49天，比如每35-49天，或每41-43天，或每42天，给予患者一次抗PD-1抗体单位剂量。
在一些实施方式中，向患者每21天施加一次1.2mg/kg-4.8mg/kg或2.4mg/kg-3.6mg/kg的抗HER2-ADC，以及100mg-200mg的抗PD-1抗体。
在一些实施方式中，向患者每21天施加一次1.2mg/kg-4.8mg/kg或2.4mg/kg-3.6mg/kg的抗HER2-ADC，以及200mg的抗PD-1抗体。
在一些实施方式中，向患者每21天施加一次1.2mg/kg-4.8mg/kg或2.4mg/kg-3.6mg/kg的抗HER2-ADC，以及向患者每42天施加一次100mg-200mg的抗PD-1抗体。
在一些实施方式中，向患者每21天施加一次1.2mg/kg-4.8mg/kg或2.4mg/kg-3.6mg/kg的抗HER2-ADC，以及向患者每42天施加一次200mg的抗PD-1抗体。
在一些实施方式中，抗HER2-ADC与抗PD-1抗体分别或同时以静脉输液形式给药。
本发明在采用抗HER2-ADC对患有HER2阳性癌症的患者进行治疗时，加入抗PD-1抗体，以提高治疗效果。在一些实施方式中，本发明的抗HER2-ADC的药效与HER2的表达水平具有正相关性，但无论HER2阳性癌症中的HER2的表达水平是高还是不高，加入抗PD-1抗体，均能提高治疗效果或降低抗HER2-ADC的有效剂量。对于HER2表达水平不高的癌症患者，在单独使用抗HER2-ADC治疗不能取得较好的治疗效果时，本发明提供的联合用药物能够对其进行治疗，拓宽了抗HER2-ADC的使用范围。
在一些实施方式中，本发明联合用药治疗的肿瘤为HER2阳性的实体瘤。
在一些实施方式中，所述实体瘤为：HER2阳性黑色素瘤、HER2阳性乳腺癌或HER2阳性胃癌。
在一些实施方式中，所述抗PD-1抗体和抗HER2-ADC在所述患者的外周血单个核细胞(PBMC)或T细胞的配合下发挥抑制肿瘤细胞增殖和/或抗肿瘤的作用。进一步地，所述抗PD-1抗体和抗HER2-ADC按照重量份数比为(1:10)-(10:1)联合用药。
在一些实施方式中，所述抗PD-1抗体和抗HER2-ADC按照重量份数比为1:1-1:10联合用药。
在一些实施方式中，所述抗PD-1抗体和抗HER2-ADC按照重量份数比为1:1-1:2联合用药。
在一些实施方式中，抗HER2-ADC为式IV化合物，与抗PD-1抗体(如抗体C)联合应用：
Figure PCTCN2020091753-appb-000016
其中，Abu为抗HER2抗体，p选自1-10，例如1、2、3、4、5、6、7、8、9或10。
在一些实施方式中，p的均值为3.3-3.7。
在一些实施方式中，Abu的轻链序列为SEQ ID NO.1，重链序列为SEQ ID NO.2；或Abu的轻链序列为SEQ ID NO.3，重链序列为SEQ ID NO.4。
在一些实施方式中，式IV的抗HER2-ADC为ADC1，其中Abu的轻链序列为SEQ ID NO.1，重链序列为SEQ ID NO.2；p的均值约为3.5。
在一些实施方式中，式IV的抗HER2-ADC为ADC2，其中Abu的轻链序列为SEQ ID NO.3，重链序列为SEQ ID NO.4；p的均值约为3.5。
在一些实施方式中，所述抗PD-1抗体为抗体C，或纳武单抗，或派姆单抗，或特瑞普利单抗，或信迪利单抗，或替雷利珠单抗，或卡瑞利珠单抗，或杰诺单抗，或者上述PD-1抗体的任意组合。
在一些实施方式中，抗PD-1抗体为抗体C，所述抗体C的轻链序列为SEQ ID NO.5，重链序列为SEQ ID NO.6。
在一些实施方式中，ADC1的单位剂量约为1.2mg/kg-6.0mg/kg；在一些实施方式中，ADC1的单位剂量约为1.2mg/kg，或2.4mg/kg，或3.6mg/kg，或4.8mg/kg，或6.0mg/kg；在一些实施方式中，ADC1的单位剂量约为4.8mg/kg。在一些实施方式中，ADC1的单位剂量约为3.6mg/kg。
在一些实施方式中，ADC1给药周期为14-28天，比如20-22天，或21天，即分别每14-28天，比如每20-22天，或每21天，给予患者一次ADC1单位剂量。
在一些实施方式中，抗体C单位剂量为约100mg-200mg。在一些实施方式中，抗体C的单位剂量约为100mg，150mg，或200mg。在一些实施方式中，抗体C的单位剂量约为1mg/kg-2mg/kg，至200mg；在一些实施方式中，抗体C的单位剂量约为2mg/kg，至200mg。
在一些实施方式中，抗体C给药周期为14-49天。在一些实施方式中，抗体C给药周期为14-28天，比如20-22天，或21天，即分别每14-28天，比如每20-22天，或每21天，给予患者一次抗体C单位剂量。在一些实施方式中，抗体C给药周期为28-49天，比如35-49天，每41-43天，或42天，给予患者一次抗体C单位剂量。
在一些实施方式中，向患者每21天施加一次1.2mg/kg-4.8mg/kg或2.4mg/kg-3.6mg/kg 的ADC1，以及100mg-200mg的抗体C。
在一些实施方式中，向患者每21天施加一次1.2mg/kg-4.8mg/kg或2.4mg/kg-3.6mg/kg的ADC1，以及200mg的抗体C。
在一些实施方式中，向患者每21天施加一次1.2mg/kg-4.8mg/kg或2.4mg/kg-3.6mg/kg的ADC1，以及每42天施加一次100mg-200mg的抗体C。
在一些实施方式中，向患者每21天施加一次1.2mg/kg-4.8mg/kg或2.4mg/kg-3.6mg/kg的ADC1，以及每42天施加一次200mg的抗体C。
在一些实施方式中，ADC1与抗体C分别或同时以静脉输液形式给药。
本发明具体实施例中使用的材料来源如下：
CCK8试剂盒：来源于Dojindo。
葡萄球菌肠毒素B(staphylococcal enterotoxin B，SEB)：军事医学科学院SL008。
细胞种类：SK-BR-3(乳腺癌细胞)，NCI-N87(胃癌细胞)，B16F10/hHER2(黑素瘤细胞)。
PBMC或T细胞：雷德生物科技有限公司。
实施例1
固定抗体C的浓度(25μg/mL)，改变ADC1的浓度(10nM起始，3倍梯度稀释)，加入PBMC或T细胞，并同时加入SEB激活PBMC或T细胞，培养72h后通过CCK法测定体外联合用药对HER2阳性和PDL1双阳性细胞的增殖抑制作用。
1、96孔板细胞铺板，SK-BR3，N87分别铺板6000个/孔和10000个/孔，每孔50μL。
2、细胞培养3-5h后加入PMBC或T细胞，PBMC与靶细胞比例为5:1，T细胞与靶细胞比例为10:1，同时加入100ng/mL的SEB，体积50μL。
3、给药：梯度稀释ADC1，10nM起始，3倍梯度稀释，共9个梯度，第10孔浓度为0。上面三排加入25μg/mL的抗体C，布板如下。
Figure PCTCN2020091753-appb-000017
4、37℃培养箱5％CO 2培养约72h，去除上清，加入含10％的CCK8的培养基。
5、37摄氏度孵育2-3h。
6、酶标仪在吸光度450读板。
结果及分析：
1、T细胞与SK-BR-3细胞10：1混合，SK-BR-3细胞1×10 4/孔。
结果如图1所示，抗体C+ADC1联用组相较ADC1单独给药组，读数明显降低，其中，抗体C+ADC1联用组中，ADC1的IC50值为0.055nM。ADC1单独给药组中，ADC1的IC50值为0.065nM。可见，在T细胞存在的情况下，抗体C的加入整体增强ADC1对SK-BR-3的抑制作用。
2、T细胞与NCI-87细胞以10：1混合
结果如图2所示，抗体C+ADC1联用组相较ADC1单独给药组，IC50值显著降低，其中，抗体C+ADC1联用组中，ADC1的IC50值为0.075nM。ADC1单独给药组中，ADC1的IC50值为0.261nM。可见，在T细胞存在的情况下，抗体C的加入整体增强ADC1对NCI-87的抑制作用。
3、PBMC与SK-BR-3细胞5：1混合，SK-BR-3细胞0.6×10 4/孔。
结果如图3所示，抗体C+ADC1联用组相较ADC1单独给药组，读数明显降低，因为ADC1存在较强的抗体依赖的细胞介导的细胞毒性作用，整体数值均偏低。可见，在PBMC存在的情况下，抗体C的加入整体增强ADC1对SK-BR-3的抑制作用。
4、对ADC1进行梯度稀释时，第10列中ADC1的浓度为0，而前三行中加入了抗体C(25μg/ml)，即第10列前3行中为单独使用抗体C的检测结果，第10列后三行中未加药，为空白对照。经对比可知，抗体C单独给药，对于SK-BR-3和NCI-87的增殖均无抑制作用。
5、SK-BR-3细胞与NCI-87细胞均为HER2高表达的细胞，实验结果表明，联用抗体C时，ADC1表现出更佳的肿瘤细胞抑制作用。ADC1和抗体C具有协同作用。
实施例2
仪器：流式细胞仪(BD Accuri C6)；台式离心机(Thermo ST16R)；生物安全柜(Antai Airtech BHC-1300IIA2)；细胞计数仪(Nexcelom Bioscience CelloMerAuto T4)；
试剂：FACS缓冲液；3％FBS(Grnco CAT#100991-148)；2mM EDTA(Grnco CAT#15575-038)；SterI Ca 2+and Mg 2+PBS(1X,Stock)；鼠Fc封闭试剂:Purified Rat Anti-Mouse CDI6/CD32(Mouse BD Fc Block TMCAT#553141)；人Fc封闭试剂:FcR Blocking Reagent,human(Miltenyi Biotec CAT#130-059-901)；PBS(Hyclone CAT#SH0256.01)；UltraComp eBeads(eBioscience CAT#01-2222-42)。
FACS抗体：hCD340(hHER2)，荧光素APC，克隆号24D2，同性对照Mouse IgG1,κ，生产商Biolegend。7-AAD，生产商BD。
FACS实验步骤：
1.用预冷FACS缓冲液洗涤细胞一次，离心、重悬细胞，细胞计数后加入FACS缓冲液调整浓度至1×10 6个1100μL。
2.转移100μL细胞至流式管，加入2μL鼠Fc封闭试剂或10μL人Fc封闭试剂分别至B16F10-hHER2或NCI-N87细胞。
3.混匀细胞，并置于4℃避光孵育10分钟。
4.加入Anti-hCD340抗体或对应的同型对照抗体(5μL/样本〉至对应的细胞孔。
5.混匀细胞，并置于4℃避光孵育30分钟。
6.加入2mL预冷FACS缓冲液至细胞孔并重悬细胞，1200rpm，4℃离心5分钟。
7.弃掉上清，加入2mL预冷FACS缓冲液至细胞孔并重悬细胞，1200rpm，4℃离心5分钟。重复一次。
8.弃掉上清，加入200μLFACS缓冲液重悬细胞，加入7-AAD(5μL/样本)至对应细胞孔，室温避光孵育10分钟，用流式细胞仪分析样本。
9.流式细胞仪产生的电子数据用Kaluza软件进行分析。
结果如图4和图5所示，统计结果如表2。可见hHER2(hCD340)在NCI-N87、B16F10-hHER2细胞株上均有表达。但B16F10-hHER2的HER2的表达强度远低于NCI-N87，说明B16F10-hHER2上的HER2并非高表达。
表2
Figure PCTCN2020091753-appb-000018
实施例3
在B16F10/hHER2细胞系中测定ADC1的IC50(9个浓度，3个复孔)。9个浓度梯度分别为240nM，48nM，9.6nM，1.92nM，0.384nM，76.8pM，15.36pM，3.07pM，0.61pM。Cisplatin作为阳性对照。
实验前细胞复苏及培养
1)用DMEM+10％FBS培养基复苏B16F10-hHER2细胞系，待细胞贴壁后16小时，使用加puromycin(嘌呤霉素)的培养基维持细胞生长至生长对数期，用膜酶进行消化，加入培养基并混匀，室温离心1000rpm 5分钟，去上清，收集细胞。加入适量培养基重悬细胞。
待测药物以及阳性对照Cisplatin对B16F10-hHER2细胞的抑制作用
第一天：细胞接种
2)取上述细胞悬液用细胞计数仪进行细胞计数：
3)根据所需测试数量进行细胞铺板，在96孔板中每孔加入100μL细胞悬液，细胞终密度为1000个/孔，每个药物浓度设定3个复孔。
4)在37℃、5％CO 2、95％湿度条件下进行细胞培养；
第二天：药物处理
待测ADC药物的储存液浓度为20mg/ml，即133330nM。用培养基进行5倍梯度稀释。
5)每孔加入50μL 3×药物溶液。待测药物最高浓度为240nM，9个浓度，5倍稀释。
6)在37℃、5％CO 2、95％湿度条件下进行细胞培养；
第五天：数据读取
7)药物处理72小时后，融化CTG试剂并平衡细胞板至室温30分钟；
8)每孔加入与培养液等体积的CTG溶液(例如:加100μLCTG/150μL细胞培养液于96孔板)；
9)在定轨摇床上振动2分钟使细胞裂解；
10)把细胞板放置于室温10分钟以稳定冷光信号；
11)用EnVision读取冷光值。
结果如图6和图7所示。其中，图6为ADC1在B16F10-hHER2细胞中的IC50曲线，计算得ADC1的IC50值为61nM。图7为阳性对照Cisplatin在B16F10-hHER2细胞中的IC50曲线，计算得Cisplatin的IC50值为38.7μM。
体外药效检测结果显示，ADC1对B16F10/hHER2增殖具有抑制作用，但IC50仅为61nM，相比ADC1对NCI-N87增殖抑制能力(IC50＝0.08nM)低700倍以上。
实施例4
一、实验方法
hPD1/hPDL1 KI HuGEMM小鼠皮下接种B16F10-hHER2细胞，建立黑色素瘤皮下移植肿瘤模型。试验分为测试药ADC1高剂量(10mg/kg，QW)、中剂量(3mg/kg，QW)和低剂量(1mg/kg，QW)，测试药抗体C(10mg/kg，BIW)，以及测试药ADC1高、中、低剂量分别与测试药抗体C联合给药。生理盐水对照组，每组6只，尾静脉注射给药，共给药两周。根据相对肿瘤抑制率(TOI)进行疗效评价，根据动物体重变化和死亡情况进行安全性评价。其中QW：指给药周期为每周一次；BIW指给药周期每周两次。给药途径为i.v(尾静脉注射)。
表3
Figure PCTCN2020091753-appb-000019
Figure PCTCN2020091753-appb-000020
注:
1.分组当天为day0，给药当天为day0。
2.联用组，先给抗体C，后给ADC1，给药间隔2小时以上。
3.试验周期(给药或者观察)之内，如果发生小鼠死亡，与药物相关死亡，需要收样，解剖做病理。
二、实验材料
1、实验动物：HPD1/hPDL1 K1 HuGEMM小鼠，雌性，6-8周(肿瘤细胞接种时的小鼠周龄)，83只小鼠用于转接。48只进组，分组小鼠体重19-26.5g。购自上海南方模式生物科技股份有限公司。饲养环境:SPF级。
2、供试品和对照品
供试品ADC1：白色微黄色冻干粉针剂，包装规格：100mg/瓶，共1瓶，4℃密封保存。
供试品抗体C：澄清或乳白色注射液，包装规格：2mg/ml/瓶，共1瓶，共4ml，4℃密封保存。
3、细胞培养
B16F10-hHER2细胞培养在含10％胎牛血清和0.5μg/ml Puromycin DMEM培养液中。收集指数生长期的B16F10-hHER2细胞，PBS重悬至适合浓度用于小鼠皮下肿瘤接种。
4、动物造模和随机分组
将收集的指数生长期的B16F10-hHER2(1×10 5)细胞重悬在0.1ml的PBS中(0.1ml/只)，于实验小鼠右侧背部皮下接种，定期观察肿瘤生长情况。
当荷瘤鼠平均肿瘤体积到达约75mm 3时，将小鼠按照表3随机分组。分组当天设定为Day 0，给药开始于Day 0(瘤体积在分组当天测量，给药时不再重复测量瘤体积)。详细的给药方法、给药剂量和给药途径见表3。
5、供试品和对照品的配制
供试品、对照品及阳性对照品溶液的配制在生物安全柜或超净工作台中进行。
给药体积＝根据体重调整给药体积(给药体积＝10μL/g)
药物配制方法：
ADC1：
第一步加入5ml注射用水与100mg ADC1复融成母液，终浓度20mg/ml。避免震荡及涡旋，母液2-8℃储存。
第二步稀释：根据母液试剂浓度20mg/ml，使用注射用水稀释后溶液在2-8℃储存不超过4h。
配置后得到溶液，现配现用，使用后丢弃。
抗体C：
根据母液实际浓度25mg/ml，使用注射用水稀释相应倍数得到给药浓度1mg/ml。稀释后溶液在2-8℃储存不超过4h。
配置后得到溶液，现配现用，使用后丢弃。
6、实验观察和数据收集
肿瘤接种后，常规监测包括了肿瘤生长及治疗对动物正常行为的影响，具体内容有实验动物的活动性，摄食和饮水情况，体重增加或降低(体重每天测量)情况，眼睛、被毛及其它异常情况。实验过程中观察到的临床症状均记录在原始数据中。肿瘤体积计算公式:肿瘤体积(mm 3)＝1/2×(a×b 2)(其中a表示长径，b表示短径)。实验中使用StudyDirector TM(版本号3.1.399.19，供应商Studylog System,Inc.,S.SanFrancisco,CA,USA)软件收集数据，包括肿瘤的长短径的测量和动物体重的称量。
当单只小鼠体重下降>20％，按照动物福利对其实施安乐死。
7、疗效评价标准
相对肿瘤增殖率，T/C％，即在某一时间点，治疗组和对照组相对肿瘤体积或瘤重的百分比值。计算公式如下:
T/C％＝T RTV/C RTV×100％(T RTV：治疗组平均RTV；C RTV：溶媒对照组平均RTV；
RTV＝V t/V 0，V 0为分组时该动物的瘤体积，V t为治疗后该动物的瘤体积)；
或T/C％＝T TW/C TW×100％(T TW：治疗组实验终结时平均瘤重；C TW：溶媒对照组实验终结时平均瘤重)。
相对肿瘤抑制率，TGI(％)，计算公式如下：TGI％＝(1-T/C)×100％。(T和C分别为治疗组和对照组在某一特定时间点的相对肿瘤体积(RTV)或瘤重(TW))。
8、统计分析
所有的统计分析和图形绘制都在R语言环境中进行(3.3.1版)。非特别说明的情况下，所有检验均为双尾检验，p值小于0.05时被认为具有统计显著性。
9、实验结果
(1)测试药ADC1和抗体C在黑色素瘤B10F16/hHER2 HuGEMM(hPD1/hPDL1 K1)模型中抗肿瘤作用研究结果
初次给药后第10天，相比较对照组(生理盐水(G01))平均肿瘤体积2719.61±508.94，治疗组测试药ADC1低剂量组(G02 1mg/kg)、中剂量组(G03 3mg/kg)和高剂量组(G04 10mg/kg)平均肿瘤体积分别为1784.89±349.96(低剂量组)、2355.02±345.36(中剂量组)和2335.15±276.99(高剂量组)，均无表显现出显著的肿瘤抑制作用(p>0.05)。测试药抗体C(G05 10mg/kg)组平均肿瘤为1587.86±470.78，TGI为38.97％，统计分析肿瘤抑制作用差异不显著(p>0.05)。测试药ADC1与测试药BAT13 06联合给药组(G06 ADC1(1mg/kg)+抗体C(10mg/kg)，G07 ADC1(3mg/kg)+抗体C(10mg/kg)，G08 ADC1(10mg/kg)+抗体C(10mg/kg))中，G08(ADC1(10mg/kg)+IBATI306(10mg/kg))平均瘤体积1292.59±314.22，TGI为 49.62％，表现出显著的肿瘤抑制作用(p<0.05)。
通过治疗组组间比较，联合给药组G08(ADC1(10mg/kg)+BAT 1306(10mg/kg))相较于ADC1高剂量组G04(10mg/kg)表现出显著地抑制肿瘤作用(p<0.05)。
肿瘤模型的HER2表达水平与ADC1的药效具有正相关性，由于B16F10/hHER2的HER2表达水平不高，因此ADC1单药的体内和体外药效并不明显。但在与抗体C联用后，表现出显著地抑制肿瘤作用。
各治疗组和对照组肿瘤生长情况见表4、表5和图8。
其中，数据以“平均值±标准误差”表示；所有组别均采用6只小鼠实验并计算平均值、标准差。
给药记录(图8箭头所示)：day0所有组；day3(抗体C BIW)：G01(vehicle)/G05/G06/G07/G08；day7所有组；day10(抗体C BIW)：G01(vehicle)G05/G06/G07/G08。
表4在动物模型中各组小鼠肿瘤体积随治疗时间的变化
Figure PCTCN2020091753-appb-000021
表5在动物模型中各组药效分析表
Figure PCTCN2020091753-appb-000022
Figure PCTCN2020091753-appb-000023
测试药ADC1和抗体C在黑色素瘤B10F16/hHER2 HuGEMM(hPD1/hPDL1 K1)模型中的安全性研究结果和讨论。
治疗组和对照组给药后体重变化见表6、图9。
其中，数据以“平均值土标准误差”表示；体重变化率与给药当天体重比较。
表6动物模型中各组体重变化情况
Figure PCTCN2020091753-appb-000024
测试药ADC1高剂量(10mg/kg，QW)、中剂量(3mg/kg，QW)和低剂量(1mg/kg，QW)，测试药抗体C(10mg/kg，BIW)，以及测试药ADC1高、中、低剂量分别与测试药抗体C联合给药，各治疗组均无动物因为明显的药物毒性而死亡，治疗期间耐受良好，ADC1与抗体C的联合应用未给小鼠带来不良反应，用药安全。
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (12)

  1. 一种抗肿瘤的联合用药物，其特征在于，包括抗HER2-抗体药物偶联物和抗PD-1抗体。
  2. 根据权利要求1所述的抗肿瘤的联合用药物，其特征在于，所述联合用药物为用于治疗HER2阳性肿瘤的联合用药物。
  3. 根据权利要求1所述的抗肿瘤的联合用药物，其特征在于，所述抗HER2-抗体药物偶联物为如式Ⅰ所示的化合物或其药学上可接受的盐，
    Figure PCTCN2020091753-appb-100001
    其中，Abu为抗HER2抗体，p选自1-10；
    X为-H或卤素基；
    Y选自-H、C1-C6烷基、C3-C6环烷基或-C(＝O)R 5；
    R 1选自-H、-OH、-OC(＝O)R 5或-OR 5基团；
    R 2为-H或C1-C6烷基；
    R 3为甲基﹑-CH 2OH或-CH 2OC(＝O)R 6；
    R 4为-OH或-SH；
    R 5为C1-C6烷基或苄基；
    R 6为C1-C6烷基﹑苯基或苄基；
    R 7为-H、C1-C6烷基或氨基酸侧链；
    R 8为-H或者C1-6烷基；
    Z独立地为-H或C1-C4烃基，或两个Z与其所连接的碳原子形成一个羰基；
    L选自任选被取代的C1-C20亚烃基或C3-C8环亚烃基，其中一个或多个-CH 2-基团独立地任选地被选自以下的基团所替代：C3-C8环亚烃基、-O-、-S-﹑-NR 8-﹑-C(＝O)-﹑-C(＝O)NR 8-﹑-NR 8C(＝O)-﹑-SO 2NR 8-或-NR 8SO 2-。
  4. 根据权利要求3所述的抗肿瘤的联合用药物，其特征在于，所述抗HER2-抗体药物偶联物为如式Ⅱ所示的化合物或其药学上可接受的盐：
    Figure PCTCN2020091753-appb-100002
    其中，Abu为抗HER2抗体，p选自1-10；
    X为-H或-Cl；
    Y为-H或甲基；
    R 7为-H、C1-C6烷基或氨基酸侧链；
    R 8为-H或者C1-C6烷基；
    L选自任选被取代的C1-C20亚烃基或C3-C8环亚烃基，其中一个或多个-CH 2-基团独立地任选地被选自以下的基团所替代：C3-C8环亚烃基、-O-、-S-﹑-NR 8-﹑-C(O)-﹑-C(＝O)NR 8-﹑-NR 8C(＝O)-﹑-SO 2NR 8-或-NR 8SO 2-。
  5. 根据权利要求4所述的抗肿瘤的联合用药物，其特征在于，所述抗HER2-抗体药物偶联物为如式Ⅲ所示的化合物或其药学上可接受的盐：
    Figure PCTCN2020091753-appb-100003
    其中，Abu为抗HER2抗体，p选自1-10；
    X为-H或-Cl；
    Y为-H或甲基；
    R 7为-H、C1-C6烷基或氨基酸侧链；
    R 8为-H或者C1-C6烷基；
    m选自1-20中的任一整数。
  6. 根据权利要求5所述的抗肿瘤的联合用药物，其特征在于，所述抗HER2-抗体药物偶 联物为如式Ⅳ所示的化合物或其药学上可接受的盐：
    Figure PCTCN2020091753-appb-100004
    其中，Abu为抗HER2抗体，p选自1-10。
  7. 根据权利要求1-6任一项所述的抗肿瘤联合用药物，其特征在于，所述抗HER2-抗体药物偶联物中的抗HER2抗体选自抗体A、抗体B、曲妥珠单抗和帕妥珠单抗中的一种或多种；和/或
    所述抗PD-1抗体为抗体C、纳武单抗、派姆单抗、特瑞普利单抗、信迪利单抗、替雷利珠单抗、卡瑞利珠单抗和杰诺单抗中的一种或多种；
    所述抗体A具有如SEQ ID NO.1所示的轻链和如SEQ ID NO.2所示的重链；
    所述抗体B具有如SEQ ID NO.3所示的轻链和如SEQ ID NO.4所示的重链；
    所述抗体C具有如SEQ ID NO.5所示的轻链和如SEQ ID NO.6所示的重链。
  8. 根据权利要求7所述的抗肿瘤的联合用药物，其特征在于，所述抗PD-1抗体和所述抗HER2-抗体药物偶联物的重量比为(1:10)-(10:1)。
  9. 根据权利要求1-7任一项所述的抗肿瘤的联合用药物，其特征在于，所述抗HER2-抗体药物偶联物与所述抗PD-1抗体分别成为独立的给药单元。
  10. 根据权利要求1-7任一项所述的抗肿瘤的联合用药物，其特征在于，所述抗HER2-抗体药物偶联物与所述抗PD-1抗体共同形成组合的给药单元。
  11. 如权利要求1-10任一项所述的抗肿瘤的联合用药物在制备抑制肿瘤细胞增殖的试剂盒、药盒、药包或药物中的应用或在制备预防和治疗肿瘤的试剂盒、药盒、药包或药物中的应用。
  12. 根据权利要求11所述的应用，其特征在于，所述肿瘤细胞为表达HER2的肿瘤细胞；和/或所述肿瘤为HER2阳性的实体瘤。
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